Self-enhancing, pharmacologically controllable expression systems

ABSTRACT

Self-enhancing, pharmacologically controllable expression systems The invention relates to a nucleic acid construct which constitutes a self-enhancing expression system and which comprises the following components:  
     at least one first structural gene that encodes an active compound;  
     at least one second structural gene that encodes a transcription factor protein; and  
     at least one activation sequence comprised of at least one sequence that binds the transcription factor protein and at least one promoter sequence;  
     wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein; and to the use of the nucleic acid construct for preparing a drug for treating diseases.

BACKGROUND OF THE INVENTION

[0001] The invention relates to a self-enhancing nucleic acid constructthat comprises at least one regulatory sequence coupled to at least onestructural gene and at least one transcription factor protein gene.

[0002] Despite various approaches taken in gene therapy, preclinical andclinical investigation results so far obtained indicate that twofundamental problems remain unsolved. One is insufficient transgenicexpression from target cells in vitro or in vivo due to intracellularshutdown processes. The second is inadequate control of transgenicexpression.

[0003] In an attempt to rectify these shortcomings of the prior art,Rivera et al. (Nature Med. 2, 1028 (1996)), Belshaw et al. (PNAS USA 93,4604 (1996)) and Ho et al. (Nature 382, 822 (1996)) have developed thefirst techniques for external control of transgenic expression. Theseapproaches are based on adding the active compound rapamycin, whichcouples two subunits together. The resulting coupling product acts as atranscription factor. The first subunit constitutes a fusion proteinformed between a DNA-binding protein and FK506-binding protein (FKBP),which protein also binds rapamycin. The second subunit is a fusionprotein which is formed between a protein FRAP, which also binds torapamycin, and the activation sequence of transcription factor proteinNF-kB.

[0004] The functional transcription factor protein that is produced bythe coupling of these two subunits with rapamycin in turn activates thesequence in the transgene for activating the structural gene.

[0005] The advantage of this external approach is that the expression ofa structural gene can be switched on or switched off by adding orremoving, respectively, the active compound rapamycin. However, thisapproach does not solve the problem of inadequate expression of astructural gene. Accordingly, the need remains for an approach toincrease transgenic expression.

SUMMARY OF THE INVENTION

[0006] The invention fulfills the unmet needs of the art by providingnucleic acid constructs, compositions containing the constructs andmethods of their use to achieve high transgenic expression. Theinvention does this by incorporating a positive feedback system withinthe nucleic acid construct itself. The resulting system is termed hereina “self-enhancing expression system.”

[0007] In one embodiment of the invention a nucleic acid construct isprovided that comprises:

[0008] at least one first structural gene that encodes an activecompound;

[0009] at least one second structural gene that encodes a transcriptionfactor protein; and

[0010] at least one activation sequence comprised of at least onesequence that binds the transcription factor protein and at least onepromoter sequence;

[0011] wherein each activation sequence activates the expression of astructural gene and the expression of the transcription factor protein.

[0012] In another embodiment of the invention a nucleic acid constructis provided that comprises:

[0013] at least one first structural gene that encodes an activecompound;

[0014] at least one second structural gene that encodes a transcriptionfactor protein; and

[0015] at least one activation sequence comprised of at least onesequence that binds said transcription factor protein and at least onepharmacological control module that comprises, in serial order, at leastone promoter,

[0016] at least one fusion protein gene coding for an activation domainof a transcription factor protein and coding for a coupling substanceprotein,

[0017] at least one promoter, at least one fusion protein gene codingfor a DNA-binding protein and coding for a second coupling substanceprotein, and at least one activation sequence that comprises a site forthe DNA-binding protein

[0018] wherein each activation sequence activates the expression of astructural gene and the expression of the transcription factor protein.

[0019] In still another embodiment of the invention a nucleic acidconstruct provided that comprises:

[0020] at least one first structural gene that encodes an activecompound;

[0021] at least one second structured gene that encodes at least onefirst fusion protein that comprises an activation domain of atranscription factor protein, and a sequence that binds a couplingsubstance;

[0022] at least one third structural gene that encodes at least onesecond fusion protein that comprises a protein that binds a couplingsubstance and a DNA-binding protein;

[0023] at least one activation sequence comprised of at least onesequence that binds said second fusion protein coupled to said firstfusion protein by a coupling substance and at least one promotorsequence;

[0024] wherein each activation sequence activates the expression of atleast one of said structural genes.

[0025] Further embodiments readily will be apparent to the skilledartisan, upon reading the specification and appended claims.

[0026] The Self-Enhancing Expression System

[0027] In its simplest embodiment, the novel self-enhancing expressionsystem comprises the following components:

[0028] a) at least one sequence a) and/or a′) for binding atranscription factor protein d),

[0029] b) at least one promoter sequence b) and/or b′),

[0030] c) at least one structural gene c) encoding an active compound,and

[0031] d) at least one gene encoding a transcription factor protein d)which binds to component a).

[0032] In conformity with the invention, components a) and/or a′) and b)and/or b′) constitute a sequence for activating transcription ofstructural gene c) and for activating expression of transcription factorprotein d).

[0033] In a preferred form according to the invention, the componentscan be arranged as depicted in FIG. 1.

[0034] The binding sequences a) and a′) can be identical or differentand bind the transcription factor d).

[0035] The promoter sequences [components b) and b′)] can be identicalor different. Low-level activation of the promoter sequences b) and b′)results in low-level expression of the structural gene [component c)]and of the gene for the transcription factor protein d) [component d)].Transcription factor protein d) made thereby, in turn binds to bindingsequences [components a) and a′)]. This binding in turn activatespromoter sequences b) and b′), bringing about an enhanced expression ofboth the structural gene and the gene for the transcription factorprotein d). This enhanced expression itself results in a higher amountof transcription factor protein d), which feeds-back and furtherstimulates this system.

[0036] According to the invention, the arrangement of the components asdepicted in FIG. 1 can be supplemented (i.e. “appended” at the upstreamend) with genes encoding a nuclear export signal (NES) and a nuclearexport factor (NEF) at the 3′ end of the structural gene. Expression ofthe NEF is under the control of an additional promoter (component b′″).This additional promoter sequence may be identical to or different fromany part of the activation sequences [components a) and b) and/or a′)and b′)] shown in FIG. 2.

[0037] The nuclear export signal (NES) is a nucleotide sequence thatimpedes the transport of a pre-messenger RNA, which is linked to it,through the nuclear membrane. The NES consequently constitutes, on itsown, a nuclear retention signal (NRS). However, if the NRS binds anexport protein, here termed “nuclear export factor” or “NEF”, the NRSgains the function of an NES. This is because the nuclear export factor(NEF) mediates the transport of the NES-containing premessenger ormessenger RNA out of the cell nucleus and into the cytoplasm. AnNES-containing premessenger or messenger RNA consequently is secretedout of the cell nucleus by its being bound to the NEF as described byFischer et al., Cell 82, 475 (1995).

[0038] In accordance with the invention, components c) and d) also canbe linked to each other (i.e. “mutually linked”) by an internal ribosomeentry site (IRES) instead of through linkage with components a′) andb′). Such IRESs lead to the expression of two DNA sequences which arelinked to each other by way of the IRES.

[0039] The linkage by way of an IRES can be effected, for example, asdepicted in FIG. 3.

[0040] This arrangement also ensures to the same extent that whenpromoter sequence b) has been subjected to low-level activation, thegene for the transcription factor protein [component d)] is alsoexpressed, by way of the IRES sequence. This expression occurs while thestructural gene [component c)] is expressed. In addition, tietranscription factor protein binds to binding sequence a), whichenhances activation of promoter sequence b), enhances expression ofstructural gene c) and, once again via the IRES sequence, also enhancesexpression of the transcription factor protein d) gene.

[0041] The arrangement, according to the invention, of individualcomponents as depicted in FIGS. 1-3 is a self-enhancing expressionsystem that operates as shown in FIG. 4. The self-enhancing expressionsystem can be extended by stringing together several identical ordifferent sequences for structural genes [components c), c′) and c″)].These structural genes are interlinked by identical or different IRESsequences, or through binding sequence a), promoter sequence b′) andpromoter sequence b″). A representative arrangement is depicted in FIG.5. The self-enhancing expression system also can be extended bystringing together several identical or different genes fortranscription factor protein d) [components d), d′) and d″)]. Onerepresentative example is shown by FIG. 6, which indicates linking byIRES sequences. The IRES sequences optionally, may be the same sequence.Binding sequence a) preferably is one type in all activation sequences.All transcription factor proteins d), d′) and d″) should bind to thisbinding sequence. When the binding sequences are not the same type (e.g.when component a is not the same as component a′), then thetranscription factor proteins [components d), d′) and d″)] should bindto all of the binding sequences. The activation sequences are preferablydesigned or selected such that they are recognized by all products oftranscription factor proteins d), d′) and d″).

[0042] In a similar manner as shown in FIG. 2, the components of FIG. 3can be supplemented with genes encoding a nuclear export signal (NES)and a nuclear export factor. This arrangement with an NES gene at the 3′end of the structural gene [component c)] and an NEF gene is shown inFIG. 7. In this last example, NEF is activated separately via anadditional promoter sequence component b′″). The component b′″ sequencecan be identical or non-identical to components a) and b).

[0043] The pharmacologically controllable promoter module In itssimplest form, the novel pharmacologically controllable promoter module(“pharmacological control module”) comprises the following components:

[0044] e) at least one promoter sequence,

[0045] f) at least one gene encoding a fusion protein f) which comprisesan activation domain of a transcription factor protein and a couplingsubstance [component j)]-binding protein A,

[0046] g) at least one further promoter sequence [identical ornon-identical to component e)] or at least one IRES,

[0047] h) at least one gene encoding a fusion protein h) which comprisesa DNA-binding domain and a coupling substance [component j)]-bindingprotein B,

[0048] i) at least one activation sequence having a site for bindingfusion protein h), and

[0049] j) at least one coupling substance j) which comprises both a siteA) for binding protein A in the fusion protein f) [expression product ofcomponent f)] and a site B) for binding protein B) in fusion protein h)[expression product of component h)].

[0050] Components e-i) can be arranged serially, as for example, inaccordance with the scheme shown in FIG. 8. This arrangement ensuresthat fusion proteins f) and h) [components f) and h)] are expressed whenpromoter sequences e) and g) are activated. When the coupling substance[component j)] is present, the two fusion proteins are linked to eachother (“mutually linked”) to form a functional transcription factorprotein for activating the activation sequence [component i)]. In thisembodiment of the invention, the promoter module, which is comprised ofindividual components, functions in the presence of a coupling substancesuch as component j). The module becomes functional upon addition of thecoupling substance component j). FIG. 9 shows an example of thisembodiment.

[0051] The Self-Enhancing, Pharmacologically Controllable ExpressionSystem

[0052] The self-enhancing expression system may be combined with apharmacologically controllable promoter module. This combination is theneffected, for example, by inserting a pharmacologically controllablepromoter module [components e) to i)] into the self-enhancing expressionsystem (see FIGS. 1, 2, 3 or 7) in place of the promoter sequence(component b) and/or b′)). The construction of this combined nucleotidesequence is illustrated, by way of example, in FIG. 10. In this example,a structural gene is transcribed only when fusion proteins f) and h)[expression products of components f) and h)] are linked by couplingsubstance component j) to form a transcription factor protein.

[0053] Alternatively, or in addition, the pharmacologically controllablepromoter module can be inserted into the self-enhancing expressionsystem in place of the gene for the transcription factor protein[component d)], the sequence for binding transcription factor protein d)[component a)] and the promoter sequence b) [component b)]. In thiscase, the binding component h) site should be attached, at least by its5′ end, to one of the promoter sequences e) and g). The construction ofthis combined nucleotide sequence is exemplified in FIG. 11.

[0054] The self-enhancing system can be combined with thepharmacologically controllable promoter module in other ways as well.For example, FIG. 8 shows that promoter sequence (component b′″)) of theNEF (see FIG. 2 or 7) can be replaced by a pharmacologicallycontrollable promoter module. One way of constructing this nucleotidesequence is shown in FIG. 12.

[0055] The nucleic acid constructs of the invention advantageouslyconsist of DNA. The term “nucleic acid construct” denotes an artificialstructure comprising nucleic acid and which can be transcribed in atarget cell. A nucleic acid construct advantageously is inserted into avector. In this context, a plasmid vector or viral vector isparticularly desirable.

[0056] Depending on the choice of promoter sequence, a novel nucleicacid construct can be used to express a structural gene [component c)]non-specifically. Alternatively, the expression is further controlled bycell specificity, virus specificity, a defined condition, or a cellcycle condition. The structural gene preferably encodes apharmacologically active compound or enzyme which cleaves an inactiveprecursor of a drug to form an active drug. The structural gene furthercan be designed to express an enzyme—ligand fusion protein. In thiscase, the ligand may bind to a cell surface. Most preferred in thiscontext is a ligand that binds to the surface of a proliferatingendothelial cell or tumour cell.

[0057] The present invention also relates to cells, particularly yeastor mammalian cells, which harbour a novel nucleic acid construct. In aparticularly desirable embodiment, a nucleic acid construct isintroduced into a cell line that is transfected by the addition oradministration of a coupling substance [component j)]. Addition of thecoupling substance stimulates expression of the structural gene. Cellsthat contain these constructions can be used to prepare a pharmaceuticalcomposition. However, the cells also can be administered to a patient asa therapeutic agent, or pharmaceutical composition, to treat a disease.Alternatively, the novel nucleic acid construct can be incorporated intoa vector and directly administered locally or parenterally into apatient. In use, the construct is designed for a particular disease suchthat the construct expresses one or more proteins, or nucleic acid thatprevents or ameliorates one or more symptoms of the disease. The amountof nucleic acid construct that is to be used in this context isdetermined by route of administration, the status of the patient, and bythe nature of the disease, as is known to a skilled artisan.

[0058] In the case of administration directly to a patient, a couplingsubstance [component j)] additionally may be administered in order toexpress the structural gene. The coupling substance causes formation ofa complete transcription factor protein by coupling fusion protein f)with fusion protein h) within transfected cells, as shown in FIG. 9.Consequently, the transfected cells only express the structural gene aslong as the coupling substance is present in the body. The duration andstrength of the expression can be controlled by administering thecoupling substance.

[0059] A preferred use of the novel nucleic acid construct consequentlyconsists in the treatment of a disease, with the provision of the drugcomprising introducing a nucleic acid construct into a target cell andexpressing the construct in a non-specific, virus-specific or targetcell-specific and/or cell cycle-specific manner by means ofadministering a coupling substance.

[0060] Nucleic acid constructs of the invention can be used to prepare apharmaceutical composition by way of synthesis in a cell. In thiscontext, a cell is transformed with a DNA construct of the invention.The transformed cell then is cultured to obtain a clone of cells,thereby creating multiple copies of the DNA construct. Preferably, geneamplification is used to increase the number of copies of the DNAconstruct in each cell. After culturing the transformed cell to obtainmultiple copies of the DNA construct, the DNA construct can be purifiedfrom the cultured cells. E. coli is preferred as the cultured cell toobtain multiple copies of the DNA construct. The purified DNA is used asa component in a pharmaceutical composition by mixing with othersubstance(s) such as buffer, salts, or other excipients as are known inthe art.

[0061] The novel nucleic acid constructs do not occur in this form innature, i.e. the structural gene for the active compound or for anenzyme or for a ligand/enzyme fusion protein is not naturally combinedwith the novel nucleic acid sequences to form a self-enhancingexpression system. This system is also not naturally combined with apharmacologically controllable promoter module.

[0062] Preferred structural genes, which are incorporated into aself-enhancing pharmacologically controllable expression system, encode10 pharmacologically active compounds. These active compounds areproteins and glycoproteins which are selected from the group consistingof cytokines, growth factors, receptor cytokines or growth factors,antibodies or antibody fragments, proteins having an antiproliferativeor cytostatic effect, angiogenesis inhibitors, thrombosis-inducingproteins, coagulation inhibitors, blood plasma proteins,complement-activating proteins, viral and bacterial coat substances,hormones, peptides having an effect on the circulation, neuropeptides,enzymes and mediators and fusion proteins which comprise at least two ofthese proteins or glycoproteins.

[0063] Detailed Description of the Components of the Self-EnhancingPharmacologically Controllable Expression System

[0064] 1) Activation Sequences and Transcription Factor Proteins forSelf-Enhancing Expression Systems

[0065] Within the meaning of the invention, the transcription factorprotein d) [gene product of component d)] binds specifically to therelevant binding sequence a) [component a)] which, for its part,activates the 3′-adjacent promoter sequence b) (or b′ or b″).

[0066] Components a) and b) consequently constitute an activationsequence which comprises a sequence for binding the relevanttranscription factor protein d).

[0067] On the other hand, the transcription factor protein d) mustcomprise a binding domain which is specific for the correspondingbinding sequence of the activator sequence [component a)], as well as atransactivation domain. An additional nuclear localization signal (NLS)promotes the interaction with the activation sequence a).

[0068] Examples of nucleic acid constructs which meet this prerequisiteare:

[0069] Embodiment A), Comprising

[0070] 1. an activation sequence comprising a component a)

[0071] having at least one sequence [e.g. nucleotide sequence:5′-CGGACAACTGTT-GACCG-3′] for binding the Gal4 protein (Chasman andKornberg, Mol. Cell Biol. 10, 2916 (1990)) and, at its 3′ end, acomponent b), which comprises:

[0072] the basal SV40 promoter

[0073]  (nucleic acids 48 to 5191; Tooze (ed.), DNA Tumor Viruses (ColdSpring Harbor N.Y., (1980), New York; Cold Spring Harbor Laboratory),

[0074] the c-fos promoter (Das et al., Nature 374, 657 (1995)) and, atits 3′ end, the HSV1 VP16 acid transactivation domain (TAD) (amino acids406 to 488; Treizenberg et al., Genes Developm. 2, 718 (1988);Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)),

[0075] the U2 sn RNA promoter and (at its 3′ end) the HSV1 VP16 TAD orat least a sequence of the activation domain of Oct-2 (amino acids 438to 479; Tanaka et al., Mol. Cell Biol. 14, 6046 (1994); Das et al.,Nature 374, 657 (1995)) or

[0076] the HSV TK promoter (Papavassiliou et al., J. Biol. Chem. 265,9402 (1990); Park et al., Molec. Endocrinol. 7, 319 (1993)) or

[0077] another non-specific, cell-specific, virus-specific or cellcycle-specific or metabolically activatable promoter, and

[0078] 2. the gene for the relevant transcription factor protein d)[component d)] containing

[0079] the cDNA for the DNA-binding domain of the Gal4 protein (aminoacids 1 to 147; Chasman and Kornberg, Mol. Cell Biol. 10, 2916 (1990)),to the 3′ end of which is attached the SV40 nuclear localization signal(NLS) (SV40 large T; amino acids 126 to 132: e.g. PKKKRKV; Dingwall etal., TIBS 16, 478 (1991)), to the 3′ end of which is attached the HSV-1VP16 acid transactivation domain (TAD) (amino acids 406 to 488;Triezenberg et al., Genes Developm. 2, 718 (1988); Triezenberg, Curr.Opin. Gen. Developm. 5, 190 (1995)).

[0080] Embodiment B) Comprising

[0081] 1. an activation sequence comprising a component a)

[0082] containing at least one sequence [e.g. nucleotide sequence5′-TACTGTATGTACA-TACAGTA-3′] for binding the LexA protein [LexAoperator; Brent et al., Nature 612, 312 (1984)] and, at its 3′ end, acomponent b) which comprises:

[0083] the SV40 basal promoter

[0084]  (nucleic acids 48 to 5191; Tooze (ed.), DNA Tumor Viruses (ColdSpring Harbor New York, (1980) New York; Cold Spring Harbor Laboratory)or another promoter (see Embodiment A), and

[0085] 2. the gene for the affiliated transcription factor protein d)[component d)] containing

[0086] the cDNA for the DNA-binding domain of the LexA protein (aminoacids 1 to 81; Kim et al., Science 255, 203 (1992)) or the whole LexAprotein (amino acids 1 to 202; Brent et al., Cell 43, 729 (1985)) to the3′ end of which is attached the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids 126 to 132: e.g. PKKKRKV; Dingwall et al.,TIBS 16, 478 (1991)), to the 3′ end of which are attached the HSV-1 VP16acid transactivation domains (TAD) (amino acids 406 to 488; Triezenberget al., Genes Developm. 2, 718 (1988); Triezenberg, Curr. Opin. Gen.Developm. 5, 190 (1995)).

[0087] Embodiment C) Comprising

[0088] 1. an activation sequence comprising a component a) containing atleast one lac operator sequence (e.g. nucleotide sequence:5′-GAATTGTGAGCGCTCACAATTC-3′) for binding the lac I repressor protein(Fuerst et al., PNAS USA 86, 2549 (1989); Simons et al., PNAS USA 81,1624 (1984)) and, at its 3′ end, a component b) which comprises:

[0089] the SV40 basal promoter (nucleic acids 48 to 5191; Tooze (ed.)DNA Tumor Viruses (Cold Spring Harbor New York, N.Y., P(1980) ColdSpring Harbor Laboratory) or another promoter (see Embodiment A), and

[0090] 2. the gene for the affiliated transcription factor protein d)[component d)] containing

[0091] the cDNA for the lac repressor (lac I) protein (Brown et al.,Cell 49, 603 (1987); Fuerst et al., PNAS USA 86, 2549 (1989)), to the 3′end of which is attached the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids: 126-132; e.g. PKKKRKV; Dingwall et al., TIBS16, 478 (1991)), to the 3′ end of which is attached the HSV-1 VP16 acidtransactivation domain (TAD) (amino acids: 406488; Triezenberg et al.,Genes Developm. 2, 718 (1988); Triezenberg, Curr. Opin. Gen. Developm.5, 190 (1995)).

[0092] Embodiment D) Comprising

[0093] 1. an activation sequence comprising a component a)

[0094] containing at least one tetracycline operator (tet O) sequence(e.g. nucleotide sequence: 5′-TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG-3′) for binding the tetracycline repressor (tet R) protein and, atits 3′ end, a component b) which comprises:

[0095] the SV40 basal promoter (nucleic acids 48 to 5191; Tooze (ed.)DNA Tumor Viruses (Cold Spring Harbor New York, (1980) N.Y., Cold SpringHarbor Laboratory) or another promoter (see Embodiment A) and

[0096] 2. the gene for the affiliated transcription factor protein d)[component d)] containing

[0097] the cDNA for the tetracycline repressor (tet R) protein (Gossenet al., PNAS USA 89, 5547 (1992); Dingermann et al., EMBO J. 11, 1487(1992)) and, at its 3′ end, the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids 126-132; e.g. PKKKRKV; Dingwall et al., TIBS16, 478 (1991)) and, at its 3′ end, the HSV-1 VP16 acid transactivationdomain (TAD) (amino acids: 406488; Triezenberg et al., Genes Developm.2, 718 (1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)).

[0098] Embodiment E) Comprising

[0099] 1. an activation sequence comprising a component a) containing atleast one sequence [e.g. nucleotide sequence 5′-TAATGATGGGCG-3′] forbinding the ZFHD-1 protein (Pomerantz et al., Science 267, 93 (1995))and, at its 3′ end, a component b) which comprises:

[0100] the basal SV40 promoter (nucleic acids 48 to 5191; Tooze (ed.),DNA Tumor Viruses (Cold Spring Harbor New York, (1980) New York, ColdSpring Harbor Laboratory) or another promoter (see Embodiment A) and

[0101] 2. the gene for the affiliated transcription factor protein d)[component d)] containing

[0102] the cDNA for the ZFHD1-protein (Pomerantz et al., Science 267, 93(1995)) and, at its 3′ end, the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids 126 to 132: e.g. PKKKRKV, Dingwall et al.,TIBS 16, 478 (1991)) and, at its 3′ end, the HSV-1 VP16 acidtransactivation domain (TAD) (amino acids 406 to 488; Triezenberg etal., Genes Developm. 2, 718 (1988); Triezenberg, Curr. Opin. Gen.Developm. 5, 190 (1995).

[0103] 2) Pharmacologically Controllable Promoter Modules

[0104] In accordance with the invention, the following genes arespecific components of the pharmacologically controllable promotermodules (see FIGS. 8 and 9 as well):

[0105] for the fusion protein f) [component f)]:

[0106] the gene for the activation domain of a transcription factorprotein, and

[0107] at least one gene for a protein A which binds the couplingsubstance j), where appropriate

[0108] supplemented with a nuclear localization signal (NLS)

[0109] for the fusion protein h) [component h)):

[0110] at least one gene for a protein B which binds the couplingsubstance j) and

[0111] the gene for a protein which binds to the DNA of the activationsequence [component i)]

[0112] for the coupling substance

[0113] the component j) having at least one site for binding the proteinA and for binding the protein B

[0114]  and the activation sequence comprising a site for binding thefusion protein h) [component h)] and a promoter element

[0115] for the component i).

[0116] The choice of the coupling substance [component j)] inevitablydetermines the nature of the component j)-binding proteins A and B incomponents f) and h), respectively. In this context, the componentj)-binding proteins A and B in components f) and h) can be identical ornon-identical. Identical component j)-binding proteins A and B can beused, in particular, when the coupling substance [component j)]possesses several identical binding sites. Within the meaning of theinvention, however, nonidentical, component j)-binding proteins A and Bare preferred in components f) and h).

[0117] This means that the coupling substance [component j)] is bound byfusion protein f) [component f)] at a site which is different from thatat which it is bound by fusion protein h) (component h)], so that fusionproteins f) and h) do not compete with each other for binding to thecoupling substance.

[0118] Coupling substances can be used which are already known to bindto particular cellular proteins whose genes can be used in components f)and h) of the pharmacologically controllable promoter.

[0119] However, this invention also relates, in particular, to usingmonoclonal antibodies, and recombinant antibodies derived therefrom, ortheir fragments, which bind to the coupling substance j). The insertionof these monoclonal antibodies, in particular their recombinant Fvfragments, into fusion proteins f) and h) [components f) and h)]constitutes a particular feature of this invention. In this context,recombinant Fv fragments which recognize different binding sites(epitopes of the A and B-binding sites, respectively) on the couplingsubstance [component j)] are preferably employed in the fusion proteins[components f) and h)].

[0120] Within the meaning of the invention, use can be made of bothmurine and human monoclonal antibodies. The murine monoclonal antibodiesare preferably employed in humanized form. The humanization is effectedin the manner described by Winter et al. (Nature 349, 293 (1991)) andHoogenbooms et al. (Rev. Tr. Transfus. Hemobiol. 36, 19 (1993)).Antibody fragments are prepared in accordance with the state of the art,for example in the manner described by Winter et al., Nature 349, 293(1993); Hoogenboom et al., Rev. Tr. Transfus. Hemobiol. 36, 19 (1993);Girol, Mol. Immunol. 28, 1379 (1991) and Huston et al., Int. Rev.Immunol. 10, 195 (1993).

[0121] Recombinant antibody fragments are prepared directly fromexisting hybridomas or are isolated from libraries of murine or humanantibody fragments (Winter et al., Annu. Rev. Immunol. 12, 433 (1994))using phage-display technology (Smith, Science 228, 1315 (1985)). Theseantibody fragments are then employed directly, at the genetic level, forfusing with other proteins or peptides [with the activation domain of atranscription factor protein (component f) or with the DNA-bindingprotein (component h)].

[0122] In order to prepare recombinant antibody fragments fromhybridomas, the genetic information which encodes the antigen-bindingdomains (VH and VL) of the antibodies is obtained by isolating the mRNA,reverse transcribing the RNA into cDNA and then amplifying the cDNA bymeans of the polymerase chain reaction (Saiki et al., Science 230, 1350(1985)) using oligonucleotides which are complementary to the 5′ and 3′ends, respectively, of the variable fragments (Orlandi et al., PNAS-USA86,3833 1989). The VH and VL fragments are then cloned into bacterialexpression vectors, for example in the form of Fv fragments (Skerra &Plückthun, Science 240, 1038 (1988), single-chain Fv fragments (scFv)(Bird et al., Science 242, 423 (1988); Huston et al., PNAS-USA 85, 5879(1988)) or Fab fragments (Better et al., Science 240, 1041 (1988)).

[0123] New antibody fragments can also be isolated directly fromantibody libraries (immune libraries or naive libraries) of murine orhuman origin using phage-display technology. In the phage display ofantibody fragments, the antigen-binding domains are cloned, either intothe phage genome (McCafferty et al., Nature 348, 552 (1990)) or intophagemid vectors (Breitling et al., Gene 104, 147 (1991)), in the formof scFv fragments (McCafferty et al., Nature 348, 552 (1990)) or Fabfragments (Hoogenboom et al., Nucl. Acid Res. 19, 4133 (1991); Barbas etal., PNAS USA 88, 7978 (1991)) as fusion proteins together with the g3Pcoat protein of filamentous bacteriophages. Antigen-binding phages areselected on antigen-loaded plastic receptacles (panning) (Marks et al.,J. Mol. Biol. 222, 581 (1991)), on antigen-conjugated, paramagneticbeads (Hawkins et al., J. Mol. Biol. 226, 889 (1992)) or by binding tocell surfaces (Marks et al., Bio/Technol. 11, 1145 (1993)).

[0124] Immune libraries are prepared by PCR amplification of thevariable antibody fragments from the B lymphocytes of immunized animals(Sastry et al., PNAS-USA 86, 5728 (1989); Ward et al., Nature 341, 544(1989); Clackson et al., Nature 352, 624 (1991)) or of patients(Mullinax et al., PNAS-USA 87, 8095 (1990); Barbas et al., PNAS-USA 88,7978 (1991)). For this, use is made of combinations of oligonucleotideswhich are specific for murine (Orlandi et al., PNAS-USA 86, 3833 (1989);Sastry et al., PNAS-USA 86, 5728 (1989)) or human immunoglobulin genes(Larrick et al., BBRC 160, 1250 (1989)), or are specific for the humanimmunoglobulin gene families (Marks et al., Eur. J. Immunol. 21, 985(1991)).

[0125] Native libraries can be prepared by using non-immunized donors asthe source of the immunoglobulin genes (Marks et al., J. Mol. Biol. 222,581 (1991)). Alternatively, immunoglobulin germ line genes can be usedto prepare semisynthetic antibody repertoires, with thecomplementarity-determining region 3 of the variable fragments beingamplified by means of PCR using degenerate primers (Hoogenboom & Winter,J. Mol. Biol. 227, 381 (1992); Barbas et al., PNAS-USA 89, 4457 (1992);Nissirn et al., EMBO J. 13, 692 (1994); Griffiths et al., EMBO J. 13,3245 (1994)). As compared with immune libraries, the so-calledsingle-pot libraries have the advantage that antibody fragments againsta large number of antigens can be isolated from one single library(Nissim et al., EMBO J. 13, 692 (1994)).

[0126] The affinity of antibody fragments can be increased still furtherusing phage-display technology, with new libraries prepared frompre-existing antibody fragments by means of random (Hawkins et al., J.Mol. Biol. 226, 889 (1992); Gram et al., PNAS-USA 89, 3576 (1992)),codon-based (Glaser et al., J. Immunol. 149, 3903 (1992)) orsite-directed mutagenesis (Balint & Larrick, Gene 137, 109 (1993), byshuffling the chains of individual domains with those of fragments fromnaive repertoires (Marks et al., Bio/Technol. 10, 779 (1992)) or byusing bacterial mutator strains (Low et al., J. Mol. Biol. 260, 359(1996)), and isolating antibody fragments having improved properties byreselecting under stringent conditions (Hawkins et al., J. Mol. Biol.226, 889 (1992)). In addition, murine antibody fragments can behumanized by the step-wise replacement of one of the variable domainswith a human repertoire and subsequent selection using the originalantigen (guided selection) (Jespers et al., Bio/Technol. 12, 889(1994)). Alternatively, murine antibodies are humanized by specificallyreplacing the hypervariable regions of human antibodies with thecorresponding regions of the original murine antibody (Jones et al.,Nature 321, 522 (1987)).

[0127] Consequently, within the meaning of the invention, the couplingsubstances basically include all substances which are able to penetrateinto a cell. Within the meaning of this invention, particular preferenceis given to those coupling substances which are already used aspharmaceuticals independently of gene therapy.

[0128] For example, these coupling substances include the followingcoupling substances [component j)] together with the affiliated proteinswhich bind to the particular coupling substance and whose genes are tobe inserted into components f) and h) in order to express the fusionproteins f) and h), respectively:

[0129] coupling substance: rapamycin or rapamycin analogues, such asL685818 (Becker et al., J. Biol. Chem. 268, 11335 (1993)), together withthe following binding proteins (and their genes);

[0130] the FK506-binding protein (FKBP; Bierer et al., Proc. Natl. Acad.Sci. USA 87, 9231, 1990))

[0131] the FKBP/raparnycin-associated protein, which binds to therapamycin/FKBP complex, or its part sequence which binds to therapamycin/FKBP complex (FRAP; Brown et al., Nature 369, 756 (1994); Chiuet al., Proc. Natl. Acad. Sci. USA 91, 12574 (1994); Sabatini et al.,Cell 78, 35 (1994); Sabers et al., J. Biol. Chem. 270, 815 (1995)).

[0132] Instead of using genes for FKBP and FRAP, use can be made ofgenes for a recombinant Fv fragment which binds to rapamycin and/orinhibits the binding of FKBP or of FRAP to rapamycin.

[0133] Coupling substance: dimers (FK1012) of FK506 (Spencer et al.,Science 262, 1019 (1993); Pruschy et al., Chem. Biol. 1, 163 (1994))together with the following binding proteins (and their genes):

[0134] the FK506-binding protein (FKBP, see above);

[0135] calcineurin (Lin et al., Cell 66, 807 (1991)) or its partsequence which binds to the FK506 complex (Clipstone et al., J. Biol.Chem. 269, 26431 (1994)); and

[0136] the gene for a recombinant Fv fragment which inhibits the bindingof FK506 to calcineurin (Ho et al., Nature 382, 822 (1996)) and can beinserted in place of the calcineurin gene.

[0137] Coupling substance: dimers of cyclosporin A

[0138]  (Belshaw et al., Proc. Natl. Acad. Sci. USA 93, 4604 (1996))together with the following binding proteins (and their genes):

[0139] cyclophilin (Belshaw et al., Proc. Natl. Acad. Sci. USA 93, 4604(1996));

[0140] calcineurin or its part sequence which binds to the cyclosporinA/cyclophilin complex (see above); and

[0141] the gene for a recombinant Fv fragment which inhibits the bindingof cyclosporin A to cyclophilin can be inserted instead of the gene forcyclophilin.

[0142] Coupling substance: monomers of cyclosporin A together with thefollowing binding proteins (and their genes):

[0143] cyclophilin;

[0144] gene for a recombinant Fv fragment which binds to cyclosporin Ain the cyclophilin/cyclosporin A complex (Cacalano et al., Molec.Immunol. 29, 107 (1992));

[0145] as an alternative to cyclophilin, use can be made of genes fordifferent recombinant Fv fragments which bind to different epitopes ofcyclosporin A (Vix et al., Proteins 15, 339 (1993); Cacalano et al.,Mol. Immunol. 29, 107 (1992); Rauffer et al., Molec. Immunol. 31, 913(1994)).

[0146] Coupling substance: methotrexate together with the followingbinding proteins (and their genes):

[0147] antibodies or antibody fragments (recombinant Fv fragments)against methotrexate (Pimm et al., Brit. J. Cancer 61, 508 (1990); Katoet al., J. Immunol. Methods 67, 321 (1984));

[0148] antibodies or antibody fragments (recombinant Fv fragments)against the pteridine group (Cot et al., Hybridoma 6, 87 (1987));

[0149] antibodies or antibody fragments (recombinant Fv fragments)against the benzene group (Cot et al., Hybridoma 6, 87 (1987)); and

[0150] dihydrofolate reductase (Masters et al., Gene 21, 59 (1983);Swift et al., Mol. Gen. Genetics 181, 441 (1981); Goldsmith et al., Mol.Cell Biol. 6, 878 (1986)).

[0151] Coupling substance: gentamycin together with the followingbinding proteins (and their genes):

[0152] antibodies or antibody fragments (recombinant Fv fragments)against gentamycin (Sierra-Madero et al., J. Clin. Microbiol. 26, 1904(1988)).

[0153] Coupling substance: ceftazidime together with the followingbinding proteins (and their genes):

[0154] antibodies or antibody fragments (recombinant Fv fragments)against ceftazidime (Shimizu et al., Int. Arch. Allergy Immunol. 98, 392(1992)).

[0155] Coupling substance: cephalexin together with the followingbinding proteins (and their genes):

[0156] antibodies or antibody fragments (recombinant Fv fragments)against the acyl side chain at the C7 position of the cephem (Nagakuraet al., Int. Arch. Allergy Applied Immunol. 93, 126 (1990)).

[0157] Coupling substance: folic acid together with the followingbinding proteins (and their genes):

[0158] folic acid-binding protein (Ratnam et al., Biochem. 28, 8249(1989); Elwood, J. Biol. Chem. 264, 14893 (1989); Sadasivan et al.,Biochem. Biophys. Acta 1131, 91 (1992))

[0159] antibodies or antibody fragments (recombinant Fv fragments)against folic acid (Rayburn et al., Clin. Chem. 30, 1007 (1984)).

[0160] Coupling substance: retinoic acid together with the followingbinding proteins (and their genes):

[0161] retinoic acid-binding domain of the cellular retinoicacid-binding protein (Stoner et al., Cancer Res. 49, 1497 (1989); Elleret al., Clin. Res. 39, 560A (1991)); and

[0162] antibodies or antibody fragments (recombinant Fv fragments)against retinoic acid (Twal et al., Developm. Biol. 168, 225 (1995);Zhou et al., J. Immunol. Methods 138, 211 (1991)).

[0163] Coupling substance: penicillin together with the followingbinding proteins (and their genes):

[0164] antibodies or antibody fragments (recombinant Fv fragments)against amoxicillin (Mayorga et al., Toxicol. 97, 225 (1995); Mayorga etal., Int. Arch. Allergy Applied Immunol. 99, 443 (1992));

[0165] antibodies or antibody fragments (recombinant Fv fragments)against the benzylpenicilloyl group (de Haan et al., Int. Arch. AllergyApplied Immunol 76, 42 (1985); Fukushima et al., Clin. Exp. Imun. 68,427 (1987));

[0166] antibodies or antibody fragments (recombinant Fv fragments)against penicillin (Sierra-Madero et al., J. Clin. Microbiol 26, 1904(1988)); and

[0167] the penicillin-binding protein (Popham et al., J. Bacteriol. 177,326 (1995); J. Bacteriol. 176, 7197 (1994)).

[0168] Coupling substance: 4-hydroxytamoxifen or tamoxifen together withthe following binding proteins (and their genes):

[0169] oestrogen-binding domain of the oestrogen receptor protein(Spreafico et al., Eur. J. Pharmacol. 227, 353 (1992); Green et al.,Nature 320 (134 (1986)); and

[0170] antibodies or antibody fragments (recombinant Fv fragments)against the oestrogen receptor/oestrogen or 4-hydroxytamoxifen complex(Giambiagi et al., J. Steroid Biochem. 30, 213 (1988); Biochim. Biophys.Acta 883, 559 (1986); Katzenellenbogen et al., Biochem. 26, 2364 (1987);Tate et al., Breast Cancer Res. Treatm. 3, 267 (1983)).

[0171] Coupling substance: tetracycline together with the followingbinding proteins (and their genes):

[0172] the tetracycline repressor protein (Gossen et al., PNAS USA 895547 (1992)); and

[0173] antibodies and antibody fragments against tetracycline.

[0174] Coupling substance: conjugate of tetracycline andisopropyl-β-D-thiogalactoside together with the following bindingproteins (and their genes):

[0175] the tetracycline repressor protein (Gossen et al., PNAS USA 89,5547 (1992)); and

[0176] the lac repressor (lac I) protein (Brow et al., Cell 49, 603(1987)).

[0177] In accordance with the invention, the genes for the couplingsubstance [component j)]-binding proteins A and B are linked

[0178] in fusion protein f) [component f), protein A], to the gene forthe activation domain of a transcription factor protein, and

[0179] in fusion protein h) [component h), protein B], to the gene for aDNA-binding protein, with this DNA-binding protein being selected suchthat it binds specifically

[0180] to the activation sequence [component i)] (see FIGS. 8 and 9). Inthis context, a “naturally occurring protein” is a protein that is foundin nature. Thus, a binding domain that comes from a “naturally occurringprotein” is distinct from a binding domain that has been designed byman. A naturally occuring protein is found and isolated from nature. Thenucleic acid sequence information for a “binding domain” may be usedseparately or combined with other sequence information to form a bindingdomain from a naturally occurring protein.

[0181] Within the meaning of the invention, the following can be used,for example, as the nucleotide sequence for the activation domain incomponent f):

[0182] herpes virus VP16 transactivation domain

[0183]  (Greaves et al., J. Virol. 64, 2716 (1990); 65, 6705 (1991));

[0184] the p65 subunit of the NF-XB transcription factor protein(Schmitz et al., EMBO J. 10, 3805 (1991)); and

[0185] the Oct-2 N-terminal glutamine-rich domain which is directly orindirectly (e.g. by way of the Gal4-binding protein) linked to the Oct-2C-terminal proline-rich Oct-2 domain (Tanaka et al., Mol. Cell Biol. 14,6046 (1994)).

[0186] The following can, for example, be used as the nucleotidesequence for the DNA-binding protein in fusion protein h), [componenth)] and as the affiliated activation sequence [component i)]:

[0187] Embodiment F), Comprising

[0188] 1. a nucleotide sequence for the DNA-binding protein in fusionprotein h), comprising:

[0189] a cDNA for the DNA-binding domain of the Gal4 protein (aminoacids 1 to 147; Chasman and Kornberg, Mol. Cell Biol. 10, 2916 (1990))and, at its 3′ end, the SV40 nuclear localization signal (NLS) (SV40large T; amino acids 126 to 132: e.g. PKKKRKV; Dingwall et al., TIBS 16,478 (1991)) and, at its 3′ end, the HSV-1 VP16 acid transactivationdomain (TAD) (amino acids 406 to 488; Triezenberg et al., GenesDevelopm. 2, 718 (1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190(1995)), and

[0190] 2. the affiliated activation sequence [component i)], comprising:

[0191] at least one sequence [e.g. nucleotide sequence5′-CGGACAACTGTTCACCG-3′] for binding the Gal4 protein (Chasman andKornberg, Mol. Cell Biol. 10, 2916 (1989)) and, at its 3′ end,

[0192] the SV40 basal promoter (nucleic acids 48 to 5191; Tooze (ed.),DNA Tumor Viruses (Cold Spring Harbor New York, N.Y.; Cold Spring HarborLaboratory), or

[0193] the c-fos promoter (Das et al., Nature 374, 657 (1995)) and, atits 3′ end, the HSV1 VP16 acid transactivation domain (TAD) (amino acids406 to 488; Triezenberg et al., Genes Developm. 2, 718 (1988);Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)), or

[0194] the U2 sn RNA promoter and, at its 3′ end, the HSV1-VP16 TAD orat least a sequence of the Oct-2 activation domain (amino acids 438 to479; Tanaka et al., Mol. Cell Biol. 14, 6046 (1994); Das et al., Nature374, 657 (1995)), or

[0195] the HSV promoter (Papavassiliou et al., J. Biol. chem. 265, 9402(1990); Park et al., Molec. Endocrinol. 7, 319 (1993)), or

[0196] any other promoter which can be activated non-specifically,cell-specifically, virus-specifically and/or cell cycle-specifically.

[0197] Embodiment G), Comprising

[0198] 1. a nucleotide sequence for the DNA-binding protein in fusionprotein h), comprising:

[0199] the cDNA for the DNA-binding domain of the LexA protein (aminoacids 1 to 81; Kim et al., Science 255, 203 (1992)) or the entire LexAprotein (amino acids 1 to 202; Brent et al., Cell 43, 729 (1985)) and,at its 3′ end, the SV40 nuclear localization signal (NLS) (SV40 large T;amino acids 126 to 132: e.g. PKKKRKV; Dingwall et al., TIBS 16, 478(1991)) and, at its 3′ end, the HSV-1 VP16 acid transactivation domains(TAD) (amino acids 406 to 488; Triezenberg et al., Genes Developm. 2,718 (1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)) and

[0200] 2. the affiliated activation sequence [component i)]:

[0201] the sequence [e.g. nucleotide sequence5′-TACTGTATGTACATACAGTA-3′] for binding the LexA protein (LexA operator,Brent et al., Nature 612, 312 (1984)], to whose 3′ end

[0202]  the SV40 basal promoter (nucleic acids 48 to 5191; Tooze (ed.),DNA Tumor Viruses (Cold Spring Harbor New York, N.Y.; Cold Spring HarborLaboratory) or another promoter (see Embodiment F) is attached.

[0203] Embodiment H), Comprising

[0204] 1. the nucleotide sequence for the DNA-binding protein in fusionprotein h), comprising

[0205] the cDNA for the lac repressor (lac I) protein (Brown et al.,Cell 49, 603 (1987); Fuerst et al., PNAS USA 86, 2549 (1989)) and, atits 3′ end, the SV40 nuclear localization signal (NLS) (SV40 large T;amino acids 126-132; e.g. PKKKRKV; Dingwall et al., TIBS 16, 478 (1991))and, at its 3′ end, the HSV-1 VP16 acid transactivation domain (TAD)(amino acids: 406-488; Triezenberg et al., Genes Developm. 2, 718(1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)), and

[0206] 2. the affiliated activation sequence [component i)]

[0207] containing at least a lac operator sequence (e.g. nucleotidesequence: 5′-GAATTGTGAGCGCTCACAATTC-3′) for binding the lac I repressorprotein (Fuerst et al., PNAS USA 86, 2549 (1989); Simons et al., PNASUSA 81, 1624 (1984)) and, at its 3′ end, the SV40 basal promoter(nucleic acids 48 to 5191; Tooze (ed.) DNA Tumor Viruses (Cold SpringHarbor New York, N.Y., Cold Spring Harbor Laboratory) or anotherpromoter (see Embodiment F).

[0208] Embodiment I), Comprising

[0209] 1. a nucleotide sequence for the DNA-binding protein in fusionprotein h), comprising

[0210] the cDNA for the tetracycline repressor (tet R) protein (Gossenet al., PNAS USA 89, 5547 (1992); Dingermann et al., EMBO J. 11, 1487(1992)) and, at its 3′ end, the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids 126-132; e.g. PKKKRKV; Dingwall et al., TIBS16, 478 (1991)) and, at its 3′ end, the HSV-1 VP16 acid transactivationdomain (TAD) (amino acids: 405-488; Triezenberg et al., Genes Developm.2, 718 (1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)),and

[0211] 2. the affiliated activation sequence [component i)]

[0212] containing at least a tetracyclin operator (tet O) sequence (e.g.nucleotide sequence: 5′-TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAA AG-3′)for binding the tetracycline repressor (tet R) protein and, at its 3′end,

[0213]  the basal SV40 promoter (nucleic acids 48 to 5191; Tooze (ed.)DNA Tumor Viruses (Cold Spring Harbor New York, N.Y., Cold Spring HarborLaboratory) or another promoter (see Embodiment F).

[0214] Embodiment J), Comprising

[0215] 1. a nucleotide sequence for the DNA-binding protein in fusionprotein h), comprising:

[0216] the cDNA for the ZFHD1 protein (Pomerantz et al., Science 267, 93(1995)) and, at its 3′ end, the SV40 nuclear localization signal (NLS)(SV40 large T; amino acids 126 to 132; e.g. PKKKRKV; Dingwall et al.,TIBS 16, 478 (1991)) and, at its 3′ end, the HSV-1 VP16 acidtransactivation domain (TAD) (amino acids 406 to 488; Triezenberg etal., Genes Developm. 2, 718 (1988); Triezenberg, Curr. Opin. gen.Developm. 5, 190 (1995)), and

[0217] 2. the affiliated activation sequence [component i)], comprising:

[0218] at least one sequence [e.g. nucleotide sequence5′-TAATGATGGGCG-3′] for binding the ZFHD-1 protein (Pomerantz et al.,Science 267, 93 (1995)) and, at its 3′ end, the basal SV40 promoter(nucleic acids 48 to 5191; Tooze (ed.), DNA Tumor Viruses (Cold SpringHarbor New York, N.Y.; Cold Spring Harbor Laboratory) or anotherpromoter (see possibility F).

[0219] 3) Promoter Sequences

[0220] Within the meaning of the invention, nucleotide sequences which,after binding transcription factor proteins, activate the transcriptionof a structural gene which is located in an adjacent position at the 3′end, are used as promoter sequences [components b), e), g) and i)]. Thechoice of the promoter sequences depends on the disease treated and onthe target cell transduced. Thus, promoter sequence can be activated inan unrestricted manner, in a target cell-specific manner, underparticular metabolic conditions, in a cell cycle-specific manner or in avirus-specific manner. Furthermore, identical or different promotersequences can be employed in components b), e) and/or g) and incomponent i). Examples of these promoter sequences, in addition to thepromoter sequences which have already been cited in Embodiments A) toJ), are:

[0221] promoters and activator sequences which can be activated in anunrestricted manner

[0222] the RNA polymerase III promoter

[0223] the RNA polymerase II promoter

[0224] the CMV promoter and CMV enhancer

[0225] the SV40 promoter

[0226] viral promoter sequences and activator sequences, such as

[0227] HBV

[0228] HCV

[0229] HSV

[0230] HPV

[0231] EBV

[0232] HTLV

[0233] HIV

[0234] When the HIV promoter is used, the entire LTR sequence includingthe TAR sequence (position ≦−453 to ≧+80, Rosen et al., Cell 41, 813(1985) is employed as the virus-specific promoter.

[0235] Promoter sequences or enhancer sequences which can be activatedmetabolically, such as an enhancer or promoter which is inducible byhypoxia.

[0236] Promoters which can be activated in a cell cycle-specific manner,such as the cdc25C gene promoter, the cyclin A gene promoter, the cdc2gene promoter, the B-myb gene promoter, the DHFR gene promoter or theE2F-1 promoter, or else binding sequences for transcription factorproteins which appear or are activated during cell proliferation. Thesebinding sequences include, for example, binding sequences forc-myc-proteins. These binding sequences also include monomers ormultimers of the nucleotide sequence which is termed a myc E box, e.g.[5′-GAAGCAGAC-CACGTGGTCTGCTTCC-3′, Blackwood and Eisenmann, Science 251,1211, (1991)].

[0237] Promoters which can be activated by tetracycline, such as thetetracycline operator in combination with a corresponding repressor.

[0238] Chimeric promoters.

[0239]  A chimeric promoter constitutes the combination of an upstreamactivator sequence, which can be activated cell-specifically,metabolically or virus-specifically, with a downstream promoter modulewhich can bind the transcription factor proteins of the CDF and CHF orE2F and CHF families and is thereby able to inhibit activation of theupstream activator sequence in the G0 and G1 phases of the cell cycle.

[0240] Hybrid promoters, for example in the form in which the TATA boxof a promoter is mutated, with this mutation being offset by acorresponding mutation in the gene of a TATA-binding protein and thisTATA-binding protein being under the control of a further promoter.

[0241] Promoters which can be activated in a cell-specific manner. Thesepromoters preferably include promoters or activator sequences from geneswhich preferably encode proteins in selected cells.

[0242] For example, within the meaning of the invention, use ispreferably to be made of promoters for the following proteins in thefollowing cells:

[0243] Promoter sequences or activator sequences which are activated inendothelial cells

[0244] brain-specific, endothelial glucose-1-transporter

[0245] endoglin

[0246] VEGF receptor 1 (flt-1)

[0247] VEGF receptor 2 (flk-1, KDR)

[0248] til-1 or til-2

[0249] B61 receptor (Eck receptor)

[0250] B61

[0251] endothelin, especially

[0252] endothelin B

[0253] endothelin-1

[0254] endothelin receptors, in particular the endothelin B receptor

[0255] IL-1α, IL-1β

[0256] IL-1 receptor

[0257] vascular cell adhesion molecule (VCAM-1)

[0258] synthetic activator sequences

[0259]  As an alternative to natural endothelial-specific promoters, usecan also be made of synthetic activator sequences which compriseoligomerized binding sites for transcription factor proteins which arepreferentially or selectively active in endothelial cells. An example ofthese transcription factor proteins is the transcription factor proteinGATA-2, whose binding site in the endothelin-1 gene, for example, is5′-TTATCT-3′.

[0260] Promoters or activator sequences which are activated in cells inthe vicinity of activated endothelial cells

[0261] VEGF

[0262]  The gene-regulatory sequences for the VEGF gene are

[0263] the 5′ flanking region, or

[0264] the 3′ flanking region, or

[0265] the c-Src gene, or

[0266] the v-Scr gene

[0267] Steroid hormone receptors and their promoter elements (Truss andBeato, Endocr. Rev. 14, 459 (1993)), in particular the mouse mammarytumour virus promoter

[0268] Promoters or activator sequences which are activated in musclecells, in particular smooth muscle cells

[0269] tropomyosin

[0270] α-actin

[0271] α-myosin

[0272] receptor for PDGF

[0273] receptor for FGF

[0274] MRF-4

[0275] phosphofructokinase A

[0276] phosphoglycerate mutase

[0277] troponin C

[0278] myogenin

[0279] receptors for endothelin A

[0280] desmin

[0281] VEGF

[0282]  The gene-regulatory sequences for the VEGF gene have alreadybeen listed in the section entitled “Promoters which are activated incells in the vicinity of activated endothelial cells” (see above).

[0283] “Artificial” promoters

[0284]  Factors of the helix-loop-helix (HLH) family (MyoD, Myf-5,myogens and MRF4) are reported to be muscle-specific transcriptionactivators. The muscle-specific transcription activators also includethe zinc finger protein GATA-4 and the MEF transcription factor groups.

[0285] The HLH proteins, and also GATA-4, exhibit muscle-specifictranscription not only with promoters of muscle-specific genes but alsoin a heterologous context, that is with artificial promoters as well.Examples of these artificial promoters are:

[0286] multiple copies of the DNA site for binding muscle-specific HLHproteins, such as the E box (Myo D) (e.g. 4×AGCAGGTGTTGGGAGGC)

[0287] multiple copies of the DNA site for binding GATA-4 of theα-myosin heavy chain genes (e.g.5′-GGCCGATGGGCAGATA-GAGGGGGCCGATGGGCAGATAGAGG3′)

[0288] Promoters and activator sequences which are activated in gliacells These include, in particular, gene-regulatory sequences orelements, respectively, from genes which, for example, encode thefollowing proteins:

[0289] the Schwann cell-specific protein periaxin

[0290] glutamine synthetase

[0291] the glia cell-specific protein

[0292]  (glial fibrillary acidic protein=GFAP)

[0293] the glia cell protein S100b

[0294] IL-6 (CNTF)

[0295] 5-HT receptors

[0296] TNFα

[0297] IL-10

[0298] insulin-like growth factor receptor I and II

[0299] VEGF

[0300]  The gene-regulatory sequences for the VEGF gene have alreadybeen listed above.

[0301] Promoters and activator sequences which are activated inhaematopoietic cells

[0302] These gene-regulatory sequences include promoter sequences forgenes for a cytokine or its receptor, which genes are expressed inhaematopoietic cells or in adjacent cells such as the stroma.

[0303] These sequences include promoter sequences for, by way ofexample, the following cytokines and their receptors:

[0304] stem cell factor receptor

[0305] stem cell factor

[0306] IL-1α

[0307] IL-1 receptor

[0308] IL-3

[0309] IL-3 receptor (α subunit)

[0310] IL-3 receptor (β subunit)

[0311] IL-6

[0312] IL-6 receptor

[0313] GM-CSF

[0314] GM-CSF receptor (α chain)

[0315] interferon regulatory factor 1 (IRF-1)

[0316]  The promoter of IRF-1 is activated to the same extent by IL-6 asby IFN-α, IFN-β or IFN-γ.

[0317] erythropoietin

[0318] erythropoietin receptor

[0319] Promoters and activator sequences which are activated inlymphocytes and/or macrophages

[0320] These include, for example, the promoter sequences and activatorsequences of the genes for cytokines, cytokine receptors and adhesionmolecules and receptors for the Fc fragment of antibodies.

[0321] Examples of these latter are:

[0322] IL-1 receptor

[0323] IL-1α

[0324] IL-1β

[0325] IL-2

[0326] IL-2 receptor

[0327] IL-3

[0328] IL-3 receptor (α subunit)

[0329] IL-3-receptor (β subunit)

[0330] IL-4

[0331] IL-4 receptor

[0332] IL-5

[0333] IL-6

[0334] interferon regulatory factor 1 (IRF-1)

[0335]  (The promoter of IRF-1 is activated to the same extent by IL-6as by IFN-α or IFN-β).

[0336] IFN-γ-responsive promoter

[0337] IL-7

[0338] IL-8

[0339] IL-10

[0340] IL-11

[0341] IFN-γ

[0342] GM-CSF

[0343] GM-CSF receptor (α chain)

[0344] IL-13

[0345] LIF

[0346] macrophage colony stimulating factor (M-CSF) receptor

[0347] type I and II scavenger macrophage receptors

[0348] MAC-1 (leukocyte function antigen)

[0349] LFA-1α (leukocyte function antigen)

[0350] p150,95 (leukocyte function antigen)

[0351] Promoter sequences and activator sequences which are activated insynovial cells

[0352] These include the promoter sequences for matrixmetalloproteinases (MMP), for example for

[0353] MMP-1 (interstitial collagenase)

[0354] MMP-3 (stroma lysin/transin)

[0355] These furthermore include the promoter sequences for tissueinhibitors of metalloproteinases (TIMP), for example

[0356] TIMP-1

[0357] TIMP-2

[0358] TIMP-3

[0359] Promoters and activator sequences which are activated inleukaemia cells

[0360] These include, for example, promoters for

[0361] c-myc

[0362] HSP-70

[0363] bcl-1/cyclin D-1

[0364] bcl-2

[0365] IL-6

[0366] 11-10

[0367] NFα, TNFβ

[0368] HOX-11

[0369] BCR-Abl

[0370] E2A-PBX-1

[0371] PML-RARA

[0372]  (promyelocytic leukaemia—retinoic acid receptor)

[0373] c-myc

[0374]  c-myc proteins bind to, and activate, multimers of thenucleotide sequence which is termed the myc E box (e.g.5′-GGAAGCAGACCACGTGGTCTGCTTCC-3′)

[0375] Promoters or activator sequences for tumour cells

[0376] A gene-regulatory nucleotide sequence with which transcriptionfactor proteins, which are formed or are active in tumour cells,interact is envisaged as the promoter sequence or activator sequence.

[0377] Within the meaning of this invention, the preferred promoters oractivator sequences include gene-regulatory sequences or elements,respectively, from genes which encode proteins which are formed, inparticular, in cancer cells or sarcoma cells. Thus, in the case ofsmall-cell bronchial carcinomas, preference is given to using thepromoter of the N-CAM protein, in the case of ovarian carcinomas tousing the promoter of the hepatitis growth factor receptor or ofL-plastin, and in the case of pancreatic carcinomas to using thepromoter of L-plastin or of polymorphic epithelial mucin (PEM).

[0378] 4) Nuclear Export Signals and Nuclear Export Factors

[0379] Within the meaning of the invention, nuclear export signals (NES)are preferably the retroviral rev-responsive element (RRE) sequences. Inthe case of HIV-1, this RRE is a sequence of 243 nucleotides(nucleotides 7362-7595; Muesing et al., Nature 313, 450 (1985)) in theenv gene (Malim et al., Nature 338, 254 (1989); Kjems et al., PNAS 88,683 (1991)). However, within the meaning of the invention, the nuclearexport signal (NES) can also be any homologous and/or functionallysimilar (analogous) nucleotide sequence such as, for example, the HBVvirus RRE-equivalent element (Huang et al., Mol. Cell Biol. 13, 7476(1993)).

[0380] In the novel nucleic acid constructs, the nuclear export factor(NEF) is a nucleotide sequence which encodes a protein which binds tothe mRNA of the NRS and mediates transport of the NRS-containingpremessenger RNA or messenger RNA out of the cell nucleus and into thecytoplasm (or out of the cytoplasm and into the cell nucleus). Withinthe meaning of the invention, use is made, in particular, of the revgene from retroviruses, especially from the HIV-1 or HIV-2 virus (Dalyet al., Nature 342, 816 (1989); Emerman et al., Cell 57, 1155 (1989);Felber et al., PNAS 86, 1495 (1989); Fischer et al., EMBO J. 13, 4105(1994)).

[0381] The rev protein of the retroviral rev gene binds, by itsN-terminal domain (Zapp et al., Nature 342, 7154 (1989); Malim et al.,Cell 65, 241 (1991)) to the RRE in the pre-mRNA (Iwai et al., Nucl.Acids Rex. 20, 6465 (1992)). The binding between the RRE and the revprotein facilitates transport of non-spliced premessenger RNA, and alsoof any other RNA which contains an RRE, out of the cell nucleus and intothe cytoplasm (Fischer et al., EMBO J. 13, 4105 (1994); Fischer et al.,Cell 82, 475 (1995)) and thereby enhances translation substantially.

[0382] Within the meaning of the invention, use can also be made, asNEF, of nucleotide sequences which encode proteins which are homologous,and functionally similar, to the HIV-1 rev protein (Bogerd et al., Cell82, 485 (1995)), such as the visna-maedi virus (VMV; Tiley et al., J.Virol. 65, 3877 (1991)) rev gene or the caprine arthritis encephalitisvirus (CAEV; Tiley et al., J. Virol. 65, 3877 (1991)) rev gene.

[0383] However, within the meaning of the invention, use can also bemade of those genes which encode proteins which, while only possessingslight, or no, homology with the rev protein are functionally similar tothe HIV-1 rev protein.

[0384] These genes include, for example, the HTLV-1 rev gene (Cullen,Microbiol. Rev. 56, 375 (1992)) and the rev gene of the equineinfectious anaemia virus (EIAV) and of the feline inrnunodeficiencyvirus (FIV) (Manusco et al., J. Virol. 68, 1988 (1994)).

[0385] In an alternative embodiment, the NEFs can also be nucleotidesequences for proteins which effect secretion of RNA out of the nucleuseven without this RNA being retained in the nucleus by means of an NRS.These proteins include, for example, the transcription factor proteinTFIIIA (Gaddat et al., Cell 60, 619 (1990); Drew et al., Gene 159, 215(1995)) or the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1protein; Pinol-Roma et al., Nature 355, 730 (1992)).

[0386] In a broader sense, the nuclear transport proteins also includeheat shock protein 70 (hsp70; Mandell et al., J. Cell Biol. 111, 1775(1990)) or the protein kinase inhibitor CPKI (Fantozzi et al., J. Biol.Chem. 269, 2676 (1994); Wen et al., J. Biol. Chem. 269, 32214 (1994)).

[0387] Features possessed in common by the NEF and its homologous andanalogous proteins are the presence of a more aminoterminally locateddomain for binding the monomeric protein to the NRS RNA (J. Virol. 64,881 (1990); Kjems et al., EMBO J. 11, 119 (1992)) and a domain which isusually leucine-rich (hnRnPA1 is an exception to this) and which isnecessary for the transport function of the NEF (Wen et al., Cell 82,463 (1995); Fischer et al., Cell 82, 475 (1995); Malim et al., J. virol.65, 4248 (1991); Venkatesh et al., Virol. 178, 327 (1990)).

[0388] Within the meaning of this invention, expression of the NEF geneis under the control of a promoter sequence [component b′″)] which islocated upstream at the 5′ end of the NEF gene (see FIGS. 2 and 7, andas already described above) or of the pharmacologically controllablepromoter module (see FIGS. 8 and 9).

[0389] 5) Internal Ribosome Entry Site (IRES)

[0390] An internal ribosome entry site makes it possible to express twoDNA sequences which are linked to each other (“mutually linked”) by wayof an IRES.

[0391] IRESs of this nature have been described, for example, byMontford and Smith TIG 11, 179 (1995); Kaufman et al., Nucl. Acids Res.19, 4485 (1991); Morgan et al., Nucl. Acids Res. 20, 1293 (1992); Dirkset al., Gene 128, 247 (1993); Pelletier and Sonenberg, Nature 334, 320(1988) and Sugitomo et al., BioTechn. 12, 694 (1994).

[0392] Thus, for example, the cDNA of the poliovirus IRES sequence(position ≦140 to ≧630 of the 5′ UTR (Pelletier and Sonenberg, Nature334, 320 (1988)) can be used to link the DNA of component c) to the DNAof component d).

[0393] 6) Structural Genes

[0394] Within the meaning of the invention, the structural genes[component c)] encode an active compound for the prophylaxis and/ortherapy of a disease. Structural genes and promoter sequences are to beselected with regard to the nature of the therapy of the disease andtaking into account the target cell to be transduced.

[0395] For example, the following combinations of promoter sequences(examples, see Section 3) and structural genes are to be selected inassociation with the following diseases:

[0396] a) Therapy of Tumours

[0397] Target cells:

[0398] proliferating endothelial cells, or

[0399] stroma cells and muscle cells which are adjacent to theendothelial cell, or

[0400] tumour cells or leukaemia cells

[0401] Promoters:

[0402] endothelial cell-specific and cell cycle-specific, or

[0403] cell non-specific or muscle cell-specific and cellcycle-specific, or

[0404] tumour cell-specific (solid tumours and leukaemias)

[0405] Structural genes for inhibitors of cell proliferation, forexample for the retinoblastoma protein (pRb=p110) or the related p107and p130 proteins

[0406] the p53 protein

[0407] the p21 (WAF-1) protein

[0408] the p16 protein

[0409] other cdk inhibitors

[0410] the GADD45 protein

[0411] the bak protein.

[0412] The retinoblastoma protein (pRb/p110) and the related p107 andp130 proteins are inactivated by phosphorylation. Preference is given tousing such genes for these cell cycle inhibitors which exhibit mutationsfor the inactivation sites of the expressed proteins without thefunction of these proteins thereby being impaired. Examples of thesemutations have been described in the case of p110.

[0413]  The DNA sequence for the p107 protein or the p130 protein ismutated in an analogous manner.

[0414] In the cell, the p53 protein is inactivated either by binding tospecial proteins, such as MDM2, or by oligomerization of the p53 by wayof the dephosphorylated C-terminal serine 392. Preference is thereforegiven to using a DNA sequence for a p53 protein which has been truncatedat the C terminus by removing the serine 392.

[0415] Structural genes for coagulation-inducing factors andangiogenesis inhibitors, for example;

[0416] plasminogen activator inhibitor-1 (PAI-1)

[0417] PAI-2

[0418] PAI-3

[0419] angiostatin

[0420] interferons, in particular

[0421] IFNα

[0422] IFNβ

[0423] IFNγ

[0424] platelet factor 4

[0425] IL-12

[0426] TIMP-1

[0427] TIMP-2

[0428] TIMP-3

[0429] leukaemia inhibitory factor (LIF)

[0430] tissue factor (TF) and its coagulation-active fragments

[0431] Structural genes for cytostatic and cytotoxic proteins, forexample for

[0432] perforin

[0433] granzyme

[0434] IL-2

[0435] IL-4

[0436] IL-12

[0437] interferons, such as

[0438] IFNα

[0439] IFNβ

[0440] IFNγ

[0441] TNF, in particular

[0442] TNFα

[0443] TNFβ

[0444] oncostatin M

[0445] sphingomyelinase

[0446] magainin and magainin derivatives

[0447] Structural genes for cytostatic or cytotoxic antibodies and forfusion proteins between antigen-binding antibody fragments andcytostatic, cytotoxic or inflammation-inducing proteins or enzymes

[0448] The cytostatic or cytotoxic antibodies include those which aredirected against membrane structures of endothelial cells, as have beendescribed, for example, by Burrows et al. (Pharmac. Ther. 64, 155(1994)), Hughes et al., (Cancer Res. 49, 6214 (1989)) and Maruyama etal., (PNAS USA 87, 5744 (1990)). These antibodies include, inparticular, antibodies against the VEGF receptors.

[0449] These antibodies furthermore include cytostatic or cytotoxicantibodies which are directed against membrane structures on tumourcells. Antibodies of this nature have been reviewed, for example, bySedlacek et al., Contrib. to Oncol. 32, Karger Verlag, Munich (1988) andContrib. to Oncol. 43, Karger Verlag, Munich (1992). Other examples areantibodies against:

[0450] sialyl Lewis

[0451] peptides on tumours which are recognized by T cells

[0452] proteins which are expressed by oncogenes

[0453] gangliosides such as GD3, GD2, GM2, 9-O-acetyl GD3, fucosyl GM1

[0454] blood group antigens and their precursors

[0455] antigens on polymorphic epithelial mucin

[0456] antigens on heat shock proteins

[0457] These antibodies furthermore include antibodies which aredirected against membrane structures of leukaemia cells. A large numberof monoclonal antibodies of this nature have already been described fordiagnostic and therapeutic methods (reviews in Kristensen, DanishMedical Bulletin 41, 52 (1994); Schranz, Therapia Hungarica 38, 3(1990); Drexler et al., Leuk. Res. 10, 279 (1986); Naeim, Dis. Markers7, 1 (1989); Stickney et al., Curr. Opin. Oncol. 4, 847 (1992); Drexleret al., Blut 57, 327 (1988); Freedman et al., Cancer Invest. 9, 69(1991)). Depending on the type of leukaemia, the following monoclonalantibodies, or their antigen-binding antibody fragments, are, forexample, suitable for use as ligands: Cells Membrane antigen Monoclonalantibodies described by AML CD13 Kaneko et al., Leuk. Lymph. 14, 219(1994) CD14 Ball, Bone Marrow Transplant. 3, 387 (1988) CD15 Campos etal., Eur. J. Cancer 28, 37 (1992) CD33 Jurcic et al., Leukaemia 9, 244(1995) CAMAL Shellard et al., Exp. Hematol. 19, 136 (1991) Sialosyl-LeMuroi et al., Blood 79, 713 (1992) B-CLL CD5 Tassone et al., Immunol.Lett. 39, 137 (1994) CD1c Orazi et al., Eur. J. Haematol. 47, CD23 28(1991) Idiotypes and isotypes Schroeder et al., Immunol. Today of themembrane 15, 289 (1994) immunoglobulins T-CLL CD33 Imai et al., J.Immunol. 151, 6470 M38 (1993) IL-2-receptors Waldmann et al., Blood 82,1701 T cell receptors (1993) ALL CALLA Morishima et al., Bone MarrowTransplant. 11, 255 (1993) CD19 Anderson et al., Blood 80, 84 (1993)Non-Hodgkin Okazaki et al., Blood 80, 84 lymphoma (1993)

[0458]  The humanization of murine antibodies, and the preparation andoptimization of the genes for Fab and recombinant Fv fragments areeffected in analogy with the methods, which have already been described,for preparing recombinant Fv fragments (see Section 2). The recombinantFv fragments are fused with genes for cytostatic, cytotoxic orinflammation-inducing proteins or enzymes in accordance with the stateof the art which is known to the skilled person.

[0459] Structural genes for fusion proteins between target cell-bindingligands and cytostatic and cytotoxic proteins.

[0460] These include all substances which bind to membrane structures ormembrane receptors on endothelial cells. For example, they include IL-1or growth factors, or their fragments or part sequences thereof, whichbind to receptors which are expressed by endothelial cells, such asPDGF, bFGF, VEGF and TGFβ (Pusztain et al., J. Pathol. 169, 191 (1993)).

[0461] They furthermore include adhesion molecules which bind toactivated and/or proliferating endothelial cells. Adhesion molecules ofthis nature, such as Slex, LFA-1, MAC-1, LECAM-1, VLA4 or vitronectin,have already been described (reviews in Augustin-Voss et al., J. CellBiol. 119, 483 (1992), Pauli et al., Cancer Metast. Rev. 2, 175 (1990),Honn et al., Cancer Metast. Rev. 11, 353 (1992) and Varner et al., CellAdh. Commun. 3, 367 (1995)).

[0462] They furthermore include substances which bind to membranestructures or membrane receptors of tumour cells or leukaemia cells. Forexample, they include growth factors, or their fragments or partsequences thereof, which bind to receptors which are expressed byleukaemia cells or tumour cells.

[0463] Growth factors of this nature have already been described(reviews in Cross et al., Cell 64, 271 (1991), Aulitzky et al., Drugs48, 667 (1994), Moore, Clin. Cancer Res. 1, 3 (1995) and Van Kooten etal., Leuk. Lymph. 12, 27 (1993)).

[0464] The genes for these ligands which bind to the target cell arefused with genes for cytostatic, cytotoxic or inflammation-inducingproteins or enzymes in accordance with the state of the art using themethods which are known to the skilled person.

[0465] Structural genes for inducers of inflammations, for example for

[0466] RANTES (MCP-2)

[0467] monocyte chemotactic and activating factor (MCAF)

[0468] IL-8

[0469] macrophage inflammatory protein-1 (MIP-1α, −β)

[0470] neutrophil activating protein-2 (NAP-2)

[0471] IL-3

[0472] IL-5

[0473] human leukaemia inhibitory factor (LIF)

[0474] IL-7

[0475] IL-11

[0476] IL-13

[0477] GM-CSF

[0478] G-CSF

[0479] M-CSF

[0480] cobra venom factor (CVF), or part sequences of CVF, whichcorrespond functionally to human complement factor C3b, i.e. which areable to bind to complement factor B and which constitute a C3 convertasefollowing cleavage by factor D.

[0481] human complement factor C3 or its part sequence C3b.

[0482] cleavage products of human complement factor C3 which resembleCVF functionally and structurally.

[0483] bacterial proteins which activate complement or elicitinflammations, such as Salmonella typhimurium porins, Staphylococcusaureus clumping factors, modulins, particularly those of Gram-negativebacteria, major outer membrane protein of legionellas or of Haemophilusinfluenza type B or of klebsiellas, or M molecules of group Gstreptococci.

[0484] Structural genes for enzymes for activating precursors ofcytostatic agents, for example for enzymes which cleave inactiveprecursor substances (prodrugs) thereby forming active cytostatic agents(drugs).

[0485] Substances of this nature, and the prodrugs and drugs with whichthey are affiliated in each case, have already been reviewed byDeonarain et al. (Br. J. Cancer 70, 786 (1994)), by Mullen (Pharmac.Ther. 63, 199 (1994)) and by Harris et al. (Gene Ther. 1, 170 (1994)).For example, the DNA sequence for one of the following enzymes is to beused:

[0486] herpes simplex virus thymidine kinase

[0487] varicella zoster virus thymidine kinase

[0488] bacterial nitroreductase

[0489] bacterial β-glucuronidase

[0490] plant β-glucuronidase from Secale cereale

[0491] human β-glucuronidase

[0492] human carboxypeptidase (CB), for example

[0493] mast cell CB-A

[0494] pancreatic CB-B

[0495] bacterial carboxypeptidase

[0496] bacterial β-lactamase

[0497] bacterial cytosine deaminase

[0498] human catalase or peroxidase

[0499] phosphatase, in particular

[0500] human alkaline phosphatase

[0501] human acid prostate phosphatase

[0502] type 5 acid phosphatase

[0503] oxidase, in particular

[0504] human lysyl oxidase

[0505] human acid D-aminooxidase

[0506] peroxidase, in particular

[0507] human glutathione peroxidase

[0508] human eosinophilic peroxidase

[0509] human thyroid peroxidase

[0510] β-galactosidase

[0511] b) Therapy of Autoimmune Diseases and Inflammations

[0512] Target cells:

[0513] proliferating endothelial cells, or

[0514] macrophages and/or lymphocytes, or

[0515] synovial cells

[0516] Promoters:

[0517] endothelial cell-specific and cell cycle-specific, or

[0518] macrophage-specific and/or lymphocyte-specific and/or cellcycle-specific

[0519] synovial cell-specific and/or cell cycle-specific

[0520] Structural genes for the therapy of allergies, for example for

[0521] IFN-β

[0522] IFN-γ

[0523] IL-10

[0524] antibodies or antibody fragments against IL-4

[0525] soluble IL-4 receptors

[0526] IL-12

[0527] TGFβ

[0528] Structural genes for preventing the rejection of transplantedorgans, for example for

[0529] IL-10

[0530] TGFβ

[0531] soluble IL-1 receptors

[0532] soluble IL-2 receptors

[0533] IL-1 receptor antagonists

[0534] soluble IL-6 receptors

[0535] immunosuppressive antibodies or their V_(H)- and V_(L)-containingfragments, or their V_(H) and V_(L) fragments which are connected by wayof a linker, which are prepared, for example, in accordance with themethod described by Marasco et al. (Proc. Natl. Acad. Sci. USA 90, 7889(1993)). Examples of immunosuppressive antibodies are antibodies

[0536] which are specific for the T cell receptor or its CD3 complex

[0537] which are directed against CD4 or CD8, and furthermore

[0538] which are directed against the IL-2 receptor, the IL-1 receptoror the IL4 receptor, or

[0539] which are directed against the adhesion molecules CD2, LFA-1,CD28 or CD40.

[0540] Structural genes for the therapy of antibody-mediated autoimmunediseases, for example for

[0541] TFGβ

[0542] IFN-α

[0543] IFN-β

[0544] IFN-γ

[0545] IL-12

[0546] soluble IL-4 receptors

[0547] soluble IL-6 receptors

[0548] immunosuppressive antibodies or their V_(H)- and V_(L)-containingfragments

[0549] Structural genes for the therapy of cell-mediated autoimmunediseases, for example for

[0550] IL-6

[0551] IL-9

[0552] IL-10

[0553] IL-13

[0554] TNFα

[0555] IL4

[0556] TNFβ

[0557] an immunosuppressive antibody or its V_(H)- and V_(L)-containingfragments

[0558] Structural genes for inhibitors of cell proliferation, cytostaticor cytotoxic proteins and enzymes for activating precursors ofcytostatic agents.

[0559] Examples of genes encoding proteins of this nature have alreadybeen cited in the section entitled “Structural genes for the therapy oftumours”.

[0560] Within the meaning of the invention, use can be made, in the sameform as already described at that point, of structural genes whichencode fusion proteins which comprise antibodies, or Fab fragments orrecombinant Fv fragments of these antibodies, or other ligands, whichare specific for the target cell, and the abovementioned cytokines,growth factors, receptors, cytostatic or cytotoxic proteins and enzymes.

[0561] Structural genes for the therapy of arthritis

[0562]  Within the meaning of the invention, structural genes areselected whose expressed protein directly or indirectly inhibitsinflammation, for example in a joint, and/or promotes the reconstitutionof extracellular matrix (cartilage and connective tissue) in the joint.

[0563] Examples of these proteins are

[0564] IL-1 receptor antagonist (IL-1-RA);

[0565]  IL-1-RA inhibits the binding of IL-1α and IL-1β

[0566] soluble IL-1 receptor;

[0567]  soluble IL-1 receptor binds and inactivates IL-1

[0568] IL-6;

[0569]  IL-6 increases the secretion of TIMP and superoxides, and

[0570] decreases the secretion of IL-1 and TNFα, by synovial cells andchondrocytes

[0571] soluble TNF receptor;

[0572]  soluble TNF receptor binds and inactivates TNF.

[0573] IL-4;

[0574]  IL-4 inhibits the formation and secretion of IL-1, TNFα and MMP

[0575] IL-10;

[0576]  IL-10 inhibits the formation and secretion of IL-1, TNFα and MMPand increases the secretion of TIMP

[0577] insulin-like growth factor (IGF-1)

[0578]  IGF-1 stimulates the synthesis of extracellular matrix.

[0579] TGFβ, especially TGFβ1 and TGFβ2

[0580]  TGFβ stimulates the synthesis of extracellular matrix.

[0581] superoxide dismutase

[0582] TIMP (tissue inhibitors of metalloproteinases)

[0583]  especially

[0584] TIMP-1

[0585] TIMP-2

[0586] TIMP-3

[0587] c) Therapy of Deficient Haematopoiesis

[0588] Target cells:

[0589] proliferating, immature cells of the haematopoietic system, or

[0590] stroma cells which are located adjacent to the haematopoieticcells

[0591] Promoters:

[0592] specific for haematopoietic cells and/or cell cycle-specific

[0593] cell non-specific

[0594] Structural genes for the therapy of anaemia, for example for

[0595] erythropoietin

[0596] Structural genes for the therapy of leucopenia, for example for

[0597] G-CSF

[0598] GM-CSF

[0599] Structural genes for the therapy of thrombocytopenia, for examplefor

[0600] IL-3

[0601] leukaemia inhibitory factor (LIF)

[0602] IL-11

[0603] thrombopoietin

[0604] d) Therapy of Nervous System Damage

[0605] Target cells:

[0606] glia cells, or

[0607] proliferating endothelial cells

[0608] Promoters:

[0609] glia cell-specific, or

[0610] endothelial cell-specific and cell cycle-specific, or

[0611] non-specific and cell cycle-specific

[0612] Structural genes for neuronal growth factors, for example FGF

[0613] nerve growth factor (NGF)

[0614] brain-derived neurotrophic factor (BDNF)

[0615] neurotrophin-3 (NT-3)

[0616] neurotrophin-4 (NT-4)

[0617] ciliary neurotrophic factor (CNTF)

[0618] Structural genes for enzymes, for example for

[0619] tyrosine hydroxylase

[0620] dopa decarboxylase

[0621] Structural genes for cytokines and their inhibitors which inhibitor neutralize the neurotoxic effect of TNFα, for example for

[0622] TGFβ

[0623] soluble TNF receptors

[0624] TNF receptors neutralize TNFα

[0625] IL-10;

[0626] IL-10 inhibits the formation of IFN gamma, TNFα, IL-2 and IL-4

[0627] soluble IL-1 receptors

[0628] IL-1 receptor I

[0629] IL-1 receptor II

[0630] Soluble IL-1 receptors neutralize the activity of IL-1

[0631] IL-1 receptor antagonist

[0632] soluble IL-6 receptors

[0633] e) Therapy of Disturbances of the Blood Coagulation System andthe Blood Circulation System

[0634] Target cells:

[0635] endothelial cells, or

[0636] proliferating endothelial cells, or

[0637] somatic cells in the vicinity of endothelial cells and smoothmuscle cells, or

[0638] macrophages

[0639] Promoters:

[0640] cell non-specific, or

[0641] cell non-specific and cell cycle-specific, or

[0642] specific for endothelial cells, smooth muscle cells ormacrophages, or

[0643] specific for endothelial cells, smooth muscle cells ormacrophages and cell cycle-specific

[0644] Structural genes for the inhibition of coagulation or for thepromotion of fibrinolysis, for example for

[0645] tissue plasminogen activator (tPA)

[0646] urokinase-type plasminogen activator (uPA)

[0647] hybrids of tPA and uPA

[0648] protein C

[0649] hirudin

[0650] serine proteinase inhibitors (serpins), such as

[0651] C-1S inhibitor

[0652] α1-antitrypsin

[0653] antithrombin III

[0654] tissue factor pathway inhibitor (TFPI)

[0655] Structural genes for promoting coagulation, for example for

[0656] F VIII

[0657] F IX

[0658] von Willebrand factor

[0659] F XIII

[0660] PAI-1

[0661] PAI-2

[0662] Structural genes for angiogenesis factors, for example for

[0663] VEGF

[0664] FGF

[0665] Structural genes for lowering blood pressure, for example for

[0666] kallikrein

[0667] endothelial cell nitric oxide synthase

[0668] Structural genes for the inhibition of the proliferation ofsmooth muscle cells following injury to the endothelial layer, forexample for

[0669] an antiproliferative, cytostatic or cytotoxic protein or for anenzyme for cleaving precursors of cytostatic agents, thereby formingcytostatic agents, as already cited above (under tumour), and fusionproteins of these active compounds together with antibodies or antibodyfragments which are specific for muscle cells.

[0670] Structural genes for other blood plasma proteins, for example for

[0671] albumin

[0672] C1 inactivator

[0673] serum cholinesterase

[0674] transferrin

[0675] α1-antitrypsin

[0676] f) Therapy of Metabolic Diseases and Genetic Disease

[0677] Target cells:

[0678] endothelial cells

[0679] muscle cells

[0680] liver cells

[0681] bronchial epithelial cells

[0682] stroma cells, or

[0683] macrophages

[0684] Promoters:

[0685] non target cell-specific, or

[0686] target cell-specific

[0687] Structural genes, for example for:

[0688] the transmembrane conductance regulator (CFTCR) in associationwith cystic fibrosis

[0689] the gene of Fanconi's anaemia

[0690] uroporphyrinogen III synthetase

[0691] iduronate 2-sulphatase (mucopolysaccharidosis type II)

[0692] β-glucuronidase (mucopolysaccharidosis VIII)

[0693] glucocerebrosidase (Gaucher's disease)

[0694] phenylalanine hydroxylase

[0695] dystrophin (Duchenne-type muscular dystrophy)

[0696] insulin receptor

[0697] human growth hormone

[0698] surfactant SP-A and SP-B-associated protein

[0699] LDL receptor

[0700] apolipoprotein B mRNA-editing protein

[0701] adenosine deaminase

[0702] g) Inoculations

[0703] Target cells:

[0704] muscle cells, or

[0705] macrophages

[0706] Promoters:

[0707] non target cell-specific, or

[0708] target cell-specific, or

[0709] target cell-specific and cell cycle-specific

[0710] Structural genes for the prophylaxis of infectious diseases

[0711] The possibilities of preparing effective vaccines conventionallyare limited (Brown, Int. J. Technol. Assessm. Health Care 10, 161(1994), Ellis, Adv. Exp. Med. Biol. 327, 263 (1992), Arnon et al., FASEBJ. 6, 3265 (1992)).

[0712] The technology of DNA vaccines was consequently developed.However, these DNA vaccines raise questions with regard to the strengthof their efficacy (Fynan et al., Int. J. Immunopharm. 17, 79 (1995);Donnelly et al., Immunol. 2, 20 (1994)).

[0713] In accordance with this invention, the self-enhancing expressionsystem increases the efficacy of the DNA vaccines.

[0714] The DNA to be selected as the active substance is the DNA for aprotein which is formed by the infectious agent and which, by means ofeliciting an immune reaction, i.e. by means of antibody binding and/orby means of cytotoxic T lymphocytes, leads to the neutralization and/ordestruction of the agent. So-called neutralization antigens of thisnature are already employed as vaccination antigens (see review inEllis, Adv. Exp. Med. Biol. 327, 263 (1992)). The following studiesprovide examples of DNA sequences which encode neutralization antigens:

[0715] influenza A virus antigen

[0716]  (Ulmer et al., Science 259, 1745 (1993), Robinson et al.,Vaccine 11, 957 (1993), Fynan et al., Int. J. Immunopharmac. 17, 79(1995))

[0717] HIV antigens

[0718]  (Wang et al., PNAS USA 90, 4156 (1993))

[0719] rabies virus antigen

[0720]  (Donnelly et al., Immunol. 2/1, 20 (1994))

[0721] HSV (herpes simplex virus) antigen

[0722]  (Fleckenstein et al., Nature 274, 57 (1978))

[0723] RSV (respiratory syncytial virus) antigen

[0724]  (Du et al., Bio/Tech. 12, 813 (1994), Hall, Science 265, 1393(1993))

[0725] parainfluenza virus antigen

[0726]  (Du et al., Bio/Techn. 12, 813 (1994))

[0727] rotavirus antigen

[0728]  (Albert et al., J. Clin. Microbiol. 25, 183 (1987), Anderson etal., J. Infect. Dis. 153, 823 (1986), Battaglia et al., J. Infect. Dis.155, 140 (1987), Chanock et al., J. Infect. Dis. 148, 49 (1983),Dyall-Smith et al., J. Virol. 38, 1099 (1981), Glass et al., Science265, 1389 (1994))

[0729] VZV (varicella zoster virus) antigen

[0730]  (Straus et al., Ann. Intern. Med. 109, 438 (1988), Gershon,Pediatr. Infect. Dis. 2, 171 (1991), Kinchington et al., J. Virol. 64,4540 (1990))

[0731] CMV (cytomegalovirus) antigen

[0732]  (Plotkin, Science 265, 1383 (1994))

[0733] measles virus antigen

[0734]  (Katz and Kellin, Science 265, 1391 (1994))

[0735] HPV (human papillomavirus) antigen

[0736]  (Tindl and Frazer, Curr. Topics Microbiol. Immunol. 186, 217(1994))

[0737] HBV (hepatitis B virus) antigen

[0738]  (Valenzuela et al., Nature 280, 815 (1979), Heerman et al., J.Virol. 52, 396 (1984))

[0739] IICV (hepatitis C virus) antigen

[0740]  (Cerny et al., Curr. Topics Microbiol. Immunol. 189, 169 (1994),Esteban et al., Progr. Liver Dis. 10, 253 (1992), Jung et al., Eur. J.Clin. Invest. 24, 641 (1994))

[0741] HDV (hepatitis D virus) antigen

[0742]  (Iwarson, Scand. J. Infect. Dis. 24, 129 (1992), Consolo et al.,Nephron. 61, 251 (1992))

[0743] HEV (hepatitis E virus) antigen

[0744]  (Iwarson, Scand. J. Infect. Dis. 24, 129 (1992), Consolo et al.,Nephron. 61, 251 (1992))

[0745] HAV (hepatitis A virus) antigen

[0746]  (d'Hondt, Vaccine 10, 48 (1992), Andre, J. Infect. Dis. 171, 33(1995), Lemon et al., Vaccine 10, 40 (1992), Melnick et al., Vaccine 10,24 (1992), Flehmig, Baillieres Clin. Gastroenterol. 4, 707 (1990))

[0747]Vibrio cholera antigen

[0748]  (Levine and Kaper, Vaccine 11, 207 (1993))

[0749]Borrelia burgdorferi antigen

[0750]  (Schaible et al., Immunol. Letters 36, 219 (1993), Wallich etal., Lab. Med. 17, 669 (1993))

[0751]Helicobacter pylori antigen

[0752]  (Crabtree et al., Lancet 338, 332 (1991), Blaser, J. Infect.Dis. 161, 626 (1990), Cover and Blaser, J. Biol. Chem. 267, 10570(1993), Cover et al., Infect. Immunol. 58, 603 (1990), Dunn et al., J.Biol. Chem. 265, 9464 (1990), Dunn et al., Infect. Immunnol. 60, 1946(1992), Lage et al., Acta Gastroenterol. Belg. 56 (suppl.), 61 (1993),Mobley et al., Scand. J. Gastroint. 26 (suppl. 187), 39 (1991))

[0753] malaria antigen

[0754]  (Nussenzweig and Long, Science 265, 1381 (1994), Maurice,Science 267, 320 (1995), Enders et al., Vaccines 10, 920 (1992), Knappet al., Infect. mm. 60, 2397 (1992)).

[0755] However, within the meaning of the invention, active substancesof this nature also include the DNA for an antiidiotype antibody, or itsantigen-binding fragments, whose antigen-binding structures (thecomplementarity-determining regions) constitute copies of the proteinstructure or carbohydrate structure of the neutralization antigen of theinfectious agent.

[0756] Antiidiotpe antibodies of this nature can, in particular, replacecarbohydrate antigens in the case of bacterial infectious agents.

[0757] Antiidiotypic antibodies of this nature, and their cleavageproducts, have been reviewed by Hawkins et al. (J. Imnnunother. 14, 273(1993)) and Westerink and Apicella (Springer Seminars in Immunopathol.15, 227 (1993)).

[0758] Structural genes for “tumour vaccines”

[0759]  These include antigens on tumour cells. Antigens of this naturehave been reviewed, for example, by Sedlacek et al., Contrib. to Oncol.32, Karger Verlag, Munich (1988) and Contrib. to Oncol 43, KargerVerlag, Munich (1992).

[0760] Other examples are constituted by the genes for the followingantigens or for anti-idiotypic antibodies corresponding to the followingantigens:

[0761] sialyl Lewis

[0762] peptides on tumours which are recognized by T cells

[0763] proteins expressed by oncogenes

[0764] blood group antigens and their precursors

[0765] antigens on polymorphic epithelial mucin and othertumour-associated mucins

[0766] antigens on heat shock proteins

[0767] gangliosides

[0768] h) The Therapy of Chronic Infectious Diseases

[0769] Target cell:

[0770] liver cell

[0771] lymphocyte and/or macrophage

[0772] epithelial cell

[0773] endothelial cell

[0774] Promoters:

[0775] virus-specific

[0776] cell-specific

[0777] virus-specific or cell-specific and cell cycle-specific

[0778] Structural genes, for example for

[0779] a protein which exhibits cytostatic or cytotoxic effects.(Examples of cytotoxic or cytostatic proteins have already been cited inthe section entitled Tumour therapy.)

[0780] an enzyme (in this regard, see the section entitled Tumourtherapy) which cleaves a precursor of an antiviral or cytotoxicsubstance, thereby forming the active substance.

[0781] Structural genes for antiviral proteins

[0782] cytokines and growth factors possessing antiviral activity.Examples of these are

[0783] IFN-α

[0784] IFN-β

[0785] IFN-γ

[0786] TNFβ

[0787] TNFα

[0788] IL-1

[0789] TGFβ

[0790] antibody having a specificity which inactivates the relevantvirus, or its V_(H)- and V_(L)-containing fragments, or its V_(H) andV_(L) fragments which are connected by way of a linker, which fragmentscan be prepared as already described in Section 2).

[0791] Examples of antibodies against viral antigens are:

[0792] anti-HBV

[0793] anti-HCV

[0794] anti-HSV

[0795] anti-HPV

[0796] anti-HIV

[0797] anti-EBV

[0798] anti-HTLV

[0799] anti-Coxsackie virus

[0800] anti-Hantaan virus

[0801] a rev-binding protein. These proteins bind to the rev RNA andinhibit rev-dependent posttranscriptional steps in retroviral geneexpression. Examples of rev-binding proteins are:

[0802] RBP9-27

[0803] RBP1-8U

[0804] RBP1-8D

[0805] pseudogenes of RBP1-8

[0806] for ribozymes which digest the MRNA of genes for cell cyclecontrol proteins or the MRNA of viruses. Ribozymes which are catalyticfor HIV have been reviewed, for example, by Christoffersen et al., J.Med. Chem. 38, 2033 (1995).

[0807] Structural genes for antibacterial proteins

[0808]  Examples of the antibacterial proteins are antibodies whichneutralize bacterial toxins or which opsonize bacteria. Examples ofthese antibodies are antibodies against

[0809] C or B meningococci

[0810]E. coli

[0811] Borrelia

[0812] Pseudomonas

[0813]Helicobacter pylori

[0814]Staphylococcus aureus

[0815] 7) Combination of Identical or Different Structural Genes

[0816] The invention furthermore relates to a self-enhancing, whereappropriate pharmacologically controllable, expression system in whichthe DNA sequences of two identical or two different structural genes[components c) and c′)] are combined. For the purpose of expressing thetwo DNA sequences, the cDNA of an internal ribosome entry site (IRES) ispreferably intercalated, as a regulatory element, between the twostructural genes.

[0817] Examples of IRES sequences of this nature have already beendescribed in Section C5).

[0818] Within the meaning of the invention, the following are examplesof preferred combinations of structural genes for

[0819] the therapy of tumours

[0820] different antiproliferative, cytostatic, cytotoxic,inflammation-inducing proteins, or

[0821] identical enzymes for cleaving the precursor of a cytostaticagent

[0822] the therapy of autoimmune diseases

[0823] different cytokines or receptors having a synergistic effect forinhibiting the cellular and/or humoral immune reaction, or

[0824] different or identical TIMPs

[0825] the therapy of deficient haematopoiesis

[0826] different, hierarchically consecutive cytokines, such as IL-1,IL-3, IL-6 or GM-CSF and erythropoietin, G-CSF or thrombopoietin

[0827] the therapy of nerve cell damage

[0828] a neuronal growth factor and a cytokine or the inhibitor of acytokine

[0829] the therapy of disturbances of the blood coagulation system andthe blood circulation system

[0830] an antithrombotic agent and a fibrinolytic agent (tPA or uPA), or

[0831] an antiproliferative, cytostatic or cytotoxic protein (or enzyme)and an antithrombotic agent or a fibrinolytic agent

[0832] vaccinations

[0833] an antigen and an immunity-stimulating cytokine, such as

[0834] IL-1α

[0835] IL-1β

[0836] IL-2

[0837] GM-CSF

[0838] IL-3, or

[0839] IL-4 receptor

[0840]  different antigens

[0841] of one infectious agent or of different infectious agents, or

[0842] of one tumour type or of different tumour types

[0843] therapy of viral infectious diseases

[0844] an antiviral protein and a cytostatic or cytotoxic protein

[0845] antibodies against different surface antigens of one virus orseveral viruses

[0846] therapy of bacterial infectious diseases

[0847] antibodies against different surface antigens and/or toxins of anorganism

[0848] 8) Insertion of Signal Sequences and Transmembrane Domains

[0849] In order to facilitate secretion of the expression product of thestructural gene, the homologous signal sequence which may be present inthe DNA sequence of the structural gene can be replaced with aheterologous signal sequence which improves extracellular secretion.

[0850] Thus, for example, the signal sequence for immunoglobulin (DNAposition ≦63 to ≧107; Riechmann et al., Nature 332, 323 (1988)) or thesignal sequence for CEA (DNA position ≦33 to ≧134; Schrewe et al., Mol.Cell Biol. 10, 2738 (1990); Berling et al., Cancer Res. 50, 6534 (1990))or the signal sequence of the human respiratory syncytial virusglycoprotein (cDNA for amino acids ≦38 to ≧50 or 48 to 65; Lichtensteinet al., J. Gen. Virol. 77, 109 (1996)) can be inserted.

[0851] Alternatively, or in addition, to the signal sequence, a sequencefor a transmembrane domain can be inserted in order to anchor the activecompound in the cell membrane of the transduced cell which is formingthe active compound.

[0852] Thus, for example, the transmembrane sequence of the humanmacrophage colony-stimulating factor (DNA position ≦1485 to ≧1554;Cosman et al., Behring Inst. Mitt. 83, 15 (1988)) or the DNA sequencefor the signal and transmembrane regions of human respiratory syncytialvirus (RSV) glycoprotein G (amino acids 1 to 63 or their part sequences,amino acids 38 to 63; Vijaya et al., Mol. Cell Biol. 8, 1709 (1988);Lichtenstein et al., J. Gen. Virol. 77, 109 (1996)), or the DNA sequencefor the signal and transmembrane regions of influenza virusneuraminidase (amino acids 7 to 35 or the part sequence amino acids 7 to27; Brown et al., J. Virol. 62, 3824 (1988)) can be inserted between thepromoter sequence and the sequence of the structural gene.

[0853] In order to enhance translation, the nucleotide sequence GCCACCor GCCGCC can, for example, be inserted at the 3′ end of the promotersequence and directly at the 5′ end of the start signal (ATG) of thesignal sequence or transmembrane sequence (Kozak, J. Cell Biol. 108, 209(1989)).

[0854] However, the nucleotide sequence for a glycophospholipid anchorcan also be inserted for the purpose of anchoring the active compound inthe cell membrane of the transduced cells which are forming the activecompound.

[0855] A glycophospholipid anchor is inserted at the 3′ end of thenucleotide sequence for the structural gene, with it being possible forthis insertion to be effected in addition to the insertion of a signalsequence.

[0856] Glycophospholipid anchors have been described, for example, forCEA (DNA position ≦893 to ≧1079; Berling et al., Cancer Res. 50, 6534(1990)), for N-CAM (Cunningham et al., Science 236, 799 (1987)) and forother membrane proteins such as Thy-1 (Clissold, Biochem. J. 281, 129(1992)) or CD16 (Selvaray et al., Nature 333, 565 (1988)).

[0857] Ferguson et al. (Ann. Rev. Biochem. 57, 285 (1988)) havepublished a review of glycophospholipid-anchored membrane proteins.

[0858] Another option for anchoring active compounds on the cellmembrane, in accordance with the present invention, is that of using aDNA sequence for a ligand/active compound fusion protein. Thespecificity of the ligand of this fusion protein is directed against amembrane structure on the cell membrane of the selected target cell.

[0859] Examples of ligands which bind to the surface of cells areantibodies or antibody fragments which are directed against structureson the surface of, for example:

[0860] endothelial cells, including, in particular, antibodies againstVEGF receptors,

[0861] or of muscle cells, such as

[0862] antibodies against actin, or

[0863] antibodies against angiotensin II receptors, or

[0864] antibodies against receptors for growth factors, such as against

[0865] EGF receptors

[0866] or against PDGF receptors

[0867] or against FGF receptors

[0868] or antibodies against endothelin A receptors

[0869] The murine monoclonal antibodies are preferably to be employed inhumanized form. Fab fragments and recombinant Fv fragments, and theirfusion products, are prepared as has already been described above.

[0870] The ligands furthermore include all active compounds, such ascytokines or adhesion molecules, growth factors, or their fragments orpart sequences thereof, or mediators, which bind to membrane structuresor membrane receptors on the particular cell selected. Examples of theseligands are ligands for endothelial cells, such as IL-1, PDGF, bFGF,VEGF, TGGβ (Pusztain et al., J. Pathol. 169, 191 (1993)) or kinin, andderivatives or analogues of kinin. The ligands furthermore includeadhesion molecules. Adhesion molecules of this nature, such as Slex,LFA-1, MAC-1, LeCAM-1, VLA-4 or vitronectin, and derivatives oranalogues of vitronectin, have already been described for endothelialcells (reviews in Augustin-Voss et al., J. Cell Biol. 119, 483 (1992);Pauli et al., Cancer Metast. Rev. 9, 175 (1990); Honn et al., CancerMetast. Rev. 11, 353 (1992); Varner et al., Cell Adh. Commun. 3, 367(1995)).

[0871] The ligands also include antibodies, or their fragments, whichare directed against tumour-specific or tumour-associated antigens onthe tumour cell membrane. Antibodies of this nature have already beendescribed in Section C6a).

[0872] The invention also relates to a composition which comprises anovel nucleic acid construct and a coupling substance j) having abinding site for the protein A of component f) and for the protein B ofcomponent h). The coupling substance j) is preferably a pharmaceuticalcomposition, in particular a substance which can penetrate through thecell membrane and into the cell, in particular rapamycin, FK506,cyclosporin A, methotrexate, folic acid, retinoic acid, penicillin,4-hydroxytamoxifen, tamoxifen or tetracycline or atetracycline/isopropyl-β-D-thiogalactoside conjugate.

[0873] The present invention also relates to cells, in particular yeastcells or mammalian cells, which harbour a novel nucleic acid constructand to a process for preparing a novel nucleic acid construct in whichthe individual components are linked to each other.

[0874] The present invention also relates to the use of a novel nucleicacid construct for preparing a drug for local, e.g. transdermal, nasal,oral, gastrointestinal, intrabronchial, intravesicular, intravaginal,intrauterine, subcutaneous, intramuscular, intradermal, periarticular orintraarticular administration, for administration into the cerebrospinalfluid, into the brain, into the liver, into the kidney, into theintestine or into the tongue, or for intraperitoneal, intrapleural orsystemic, e.g. intravenous, intraarterial, intraportal or intracardial,administration for the prophylaxis and/or therapy of tumours,leukaemias, automimmune diseases, inflammations, damage to the nervoussystem, disturbances of the blood coagulation system and bloodcirculatory system, metabolic diseases, genetic damage, viral orbacterial infectious diseases and/or deficient haematopoiesis and/or forvaccinating against viral, bacterial or parasitic infections and/oragainst tumours, and to the use of a cell according to the invention forpreparing a drug for local or systemic administration for theprophylaxis and/or therapy of diseases.

DESCRIPTION OF THE FIGURES

[0875]FIG. 1 shows a diagram of the novel nucleic acid construct in itssimplest form.

[0876]FIG. 2 shows a diagram of the nucleic acid construct depicted inFIG. 1 after having been supplemented with the gene encoding a nuclearexport signal (NES) and the gene encoding a nuclear export factor (NEF).

[0877]FIG. 3 shows a diagram of the linking of components c) and d) byway of an IRES.

[0878]FIG. 4 shows a diagram of the scheme by which the individualcomponents depicted in FIGS. 1-3 react.

[0879]FIG. 5 shows a diagram of the enlargment of the nucleic acidconstruct with additional structural genes.

[0880]FIG. 6 shows a diagram of the enlargement of the nucleic acidconstruct with additional genes for the transcription factor protein.

[0881]FIG. 7 shows a diagram of the nucleic acid construct depicted inFIG. 3 following supplementation with the genes encoding NES and NEF.

[0882]FIG. 8 shows a diagram of a pharmacologically controllablepromoter module in its simplest form.

[0883]FIG. 9 shows a diagram of the scheme by which the individualcomponents depicted in FIG. 8 react.

[0884]FIG. 10 shows a diagram of a self-enhancing, pharmacologicallycontrollable expression system.

[0885]FIG. 11 shows a diagram of the replacement of components a) and b)with component i) and the replacement of component d), together with theIRES region, with the pharmaceutically controllable promoter modulecontaining components e), f), g) and h).

[0886]FIG. 12 shows a diagram of the replacement of components b′″) withthe pharmaceutically controllable promoter module containing componentse), f), g), h) and i).

[0887]FIG. 13 shows a diagram of a self-enhancing expression system forthe cell cycle-specific and cell-specific expression of β-glucuronidase.

[0888]FIGS. 14 and 15 shows diagrams of two expression systems which arenot self-enhancing.

[0889]FIG. 16 shows a diagram of a self-enhancing, pharmacologicallycontrollable expression system for the cell cycle-specific andpharmacologically controllable expression of β-glucuronidase.

[0890] The invention is explained in more detail with the aid of thefollowing examples.

EXAMPLES

[0891] 1. Construction of a Self-Enhancing Expression System

[0892] A self-enhancing expression system in accordance with the schemedepicted in FIG. 13 is prepared for the purpose of expressingβ-glucuronidase in a cell cycle-specific and cell-specific manner.

[0893] The DNA sequences of the individual components are joinedtogether, in the 5′ to 3′ direction, as follows:

[0894] component a):

[0895] the sequence [nucleotide sequence: 5′-CGGACAACTGTTGACCG-3′] forbinding the Gal4 protein (Chasman and Kornberg, Mol. Cell Biol. 10, 2916(1990))

[0896] component b):

[0897] the promoter sequence of the cdc25C gene [nucleic acids: −290 to+121; Lucibello et al., EMBO J. 14, 132 (1995); Zwicker at al., Nucl.Acids Res. 23, 3822 (1995); EMBO J. 14, 4514 (1995))

[0898] component c):

[0899] the sequence GCCACC (Kodak, J. Cell Biol. 108, 229 (1989))

[0900] the cDNA for the immunoglobulin signal peptide [nucleotidesequence ≦63 to ≧107; Riechmann et al., Nature 332, 323 (1988))

[0901] the cDNA for human β-glucuronidase [nucleotide sequence ≦93 to≧1982; Oshima et al., PNAS USA 84, 685 (1987))

[0902] component a′):

[0903] the sequence for binding the Gal4 protein (Chasman and Kornberg,Mol. Cell Biol. 10, 2916 (1990))

[0904] component b′):

[0905] the promoter sequence of the VEGF receptor gene [nucleic acids−1195 to +>100; Morishita et al., J. Biol. Chem. 270, 27948 (1995))

[0906] component d):

[0907] the cDNA for the DNA-binding domain of the Gal4 protein [aminoacids 1 to 147; Chasman and Kornberg, Mol. Cell Biol. 10, 2916 (1990))

[0908] the cDNA for the SV40 nuclear localization signal (NLS) (SV40large T; amino acids 126 to 132: PKKKRKV; Dingwall et al., TIBS 16, 478(1991))

[0909] the cDNA for the HSV-1 VP16 acid transactivation domain (TAD)[amino acids 406 to 488; Triezenberg et al., Genes Developm. 2, 718(1988); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995))

[0910] The individual components of the construct are linked by way ofsuitable restriction sites which are concomitantly introduced at thetermini of the different elements during PCR amplification. Thecomponents are linked using enzymes, which are specific for therestriction sites, and DNA ligases which are known to the skilledperson. These enzymes can be obtained commercially.

[0911] Human umbilical cord endothelial cells and fibroblasts (Wi-38)which are being maintained in culture are transfected with the describedplasmid using the method known to the skilled person (Lucibello et al.,EMBO J. 14, 132 (1995)), and the quantity of β-glucuronidase which isproduced by the endothelial cells is measured using4-methylumbelliferyl-β-glucuronide as the substrate.

[0912] For the purpose of checking the cell-cycle specificity,endothelial cells are synchronized in G0/G1 by removing methionine for48 hours. The DNA content of the cells is determined in afluorescence-activated cell sorter following staining with Hoechst 33258(Hoechst A G, Frankfurt) (Lucibello et al., EMBO J. 14, 132 (1995)).

[0913] The following results are obtained:

[0914] It is not possible to detect any increase in β-glucuronidase intransfected fibroblasts as compared with untransfected fibroblasts.

[0915] Transfected endothelial cells express markedly moreβ-glucuronidase than do untransfected endothelial cells.

[0916] Proliferating endothelial cells (DNA>2S) secrete markedly moreβ-glucuronidase than do endothelial cells which are synchronized inG0/G1 (DNA =2S).

[0917] Consequently, the self-enhancing expression system which has beendescribed leads to a cell-specific, cell cycle-dependent expression ofthe structural gene β-glucuronidase.

[0918] The strength of the expression due to the novel self-enhancingexpression system is now compared with that due to two expressionsystems which are not self-enhancing. These latter systems are preparedin accordance with the schemes depicted in FIGS. 14 and 15.

[0919] In this case, the constituents of components b), b′) and c) areidentical, as already described for the scheme depicted in FIG. 13.

[0920] The individual components of the construct are linked by way ofsuitable restriction sites, which are concomitantly introduced at thetermini of the different elements during PCR amplification. Thecomponents are linked using enzymes, which are specific for therestriction sites, and DNA ligases which are known to the skilledperson. These enzymes can be obtained commercially.

[0921] The nucleotide construct which has been prepared in this way iscloned into pUC18/19 or Bluescript-derived plasmid vectors.

[0922] Human umbilical cord endothelial cells which are being maintainedin culture are transfected with the described plasmids using the methodknown to the skilled person (Lucibello et al., EMBO J. 14, 132 (1995)),and the quantity of β-glucuronidase which is produced by the endothelialcells is measured using 4-methylumbelliferyl-β-glucuronide as thesubstrate.

[0923] The following results are obtained:

[0924] Proliferating endothelial cells which are transfected with theplasmid comprising the nucleic acid construct depicted in FIGS. 14) and15) express markedly less β-glucuronidase than do proliferatingendothelial cells which are transfected with the plasmid comprising thenovel nucleic acid construct depicted in FIG. 13).

[0925] The self-enhancing expression system which has been describedgives rise to markedly enhanced expression of the structural geneβ-glucuronidase.

[0926] 2. Construction of a Self-Enhancing, PharmacologicallyControllable Expression System

[0927] A self-enhancing, pharmacologically controllable expressionsystem, corresponding to the structure shown in the diagram in FIG. 11,is prepared for the cell cycle-specific and pharmacologicallycontrollable expression of β-glucuronidase, as depicted in FIG. 16.

[0928] The DNA sequences of the individual components are joinedtogether, in the 5′ to 3′ direction, as follows:

[0929] component i):

[0930] the sequence [nucleotide sequence: 5′-CGGACAACTGTT(3ACCG-3′] forbinding the Gal4 protein (Chasman and Kornberg, Mol. Cell Biol. 10, 2916(1990))

[0931] the SV40 basal promoter [nucleotides 48 to 5191; Tooze (ed.), DNATumor Viruses (Cold Spring Harbor New York, N.Y., Cold Spring HarborLaboratory)]

[0932] component c):

[0933] the sequence GCCACC (Kodak, J. Cell Biol. 108, 229 (1989))

[0934] the cDNA for the immunoglobulin signal peptide [nucleotidesequence ≦63 to ≧107; Riechmann et al., Nature 332, 323 (1988)]

[0935] the cDNA for human β-glucuronidase [nucleotide sequence ≦93 to≧1982; Oshima et al., PNAS USA 84, 685 (1987)]

[0936] component e):

[0937] the sequence for binding the Gal protein (Chasman and Kornberg,Mol. Cell Biol. 10, 2916 (1990))

[0938] the promoter sequence of the cdc25C gene [nucleotides −290 to+121; Lucibello et al., EMBO J. 14, 132 (1995); Zwicker at al., Nucl.Acids Res. 23, 3822 (1995); EMBO J. 14, 4514 (1995)]

[0939] component f):

[0940] the cDNA for the herpes virus VP16 activation domain (Greaves etal., J. Virol. 64, 2716 (1990); 65, 6705 (1991))

[0941] the cDNA for recombinant anti-cyclosporin A Fv (A) (protein A)

[0942] component g):

[0943] corresponds to component e)

[0944] component h):

[0945] the cDNA for recombinant anti-cyclosporin A Fv (B) (protein B)

[0946] the cDNA for the DNA-binding domain of the Gal4 protein [aminoacids 1 to 147; Chasman and Kornberg, Mol. Cell Biol. 10, 2916 (1990)]

[0947] the cDNA for the SV40 nuclear localization signal (NLS) [SV40large T; amino acids 126 to 132: PKKKRKV; Dingwall et al., TIBS 16, 478(1991)]

[0948] the cDNA for the HSV-1 VP16 acid transactivatlon domain (TAD)[amino acids 406 to 488; Triezenberg et al., Genes Developm. 2, 718(1998); Triezenberg, Curr. Opin. Gen. Developm. 5, 190 (1995)].

[0949] Antibodies against cyclosporin A are prepared as described byCacalano et al., Mol. Immunol. 29, 107 (1992). For this, cyclosporin Ais coupled to bovine serum albumin using 4-benzoylbenzoic acid and UVlight. The coupling product is administered several times subcutaneouslyto Balb/c mice. The spleen cells are isolated 14 days after the lastimmunization. The mRNA is extracted from these cells using an mRNAextraction kit (from Pharmacia, Freiburg). Reverse transcription is thenused to transcribe this mRNA into cDNA with the aid of a cDNA synthesiskit and random hexaoligonucleotides (from Pharmacia, Freiburg). ThiscDNA serves as the starting material for amplifying the variable heavychain, or the variable light chain, of the immunoglobulins by means ofthe polymerase chain reaction (Saiki et al., Science 230, 1350 (1985))using specific primers (Clackson et al., Nature 352, 624 (1991); Sastryet al., PNAS USA 86, 5728 (1989); Ward et al., Nature 341, 544 (1989);Orlandi et al., PNAS USA 86, 3833 (1989)).

[0950] The primers are used to, at the same time, introduce restrictioncleavage sites for cloning the fragments into the bacterial expressionvector pHENIS (which is derived from pHEN™; Hoogenboom et al., Nucl.Acids Res. 19, 4133 (1991); see FIG. 1)). This vector comprises a pelBsignal sequence for periplasmic secretion, a myc tag for detection withthe monoclonal antibody 9E10, a histidine tag for purification by meansof immobilized metal affinity chromatography (IMAC), as well as acloning region for the heavy chain and the light chain and a shortsequence which encodes a glycine-serine linker of 14 amino acids inlength. Fusion with the gene3 protein (g3P) is also effected for thepurpose of displaying on the surface of bacteriophages. The heavy andlight chains are digested with the appropriate restriction enzymes (VIIwith SfiI and Shol; VL with ApaLI and Notl) and cloned consecutivelyinto the vector. This results in a recombinant single-chain Fv fragmentcomprising the variable heavy chain and variable light chain, which arelinked covalently by means of a short peptide sequence.

[0951] In conformity with the phage-display of antibody fragments, theantigen-binding domains are cloned in the form of scFv fragments(McCafferty et al., Nature 348, 552 (1990)), as fusion proteins with thefilamentous bacteriophage coat protein g3P, into phagemid vectors(Breitling et al., Gene 104 147 (1994)). Antigen-binding phages areselected on cyclosporin A-loaded plastic receptacles (panning) (Marks etal., J. Mol. Biol. 222, 581 (1991)).

[0952] Phages which bind to cyclosporin A are cloned and multiplied andthen once again selected on cyclosporin A-loaded plastic receptacles.After selecting four times, two clones (proteins A and B) are chosenwhich do not inhibit each other's binding to cyclosporin A.

[0953] The murine recombinant Fv fragments (proteins A and B) arehumanized by specifically replacing the hypervariable regions of humanantibodies with the corresponding regions of this murine recombinant Fvfragment (Jones et al., Nature 321, 522 (1987)).

[0954] The construct is linked by way of suitable restriction sites,which are introduced at the termini of the different elements during PCRamplification. The linking is effected using enzymes, which are specificfor the restriction sites, and DNA ligases which are known to theskilled person. These enzymes can be obtained commercially.

[0955] The nucleotide construct which has been prepared in this way iscloned into pUC18/19-derived or Bluescript-derived plasmid vectors.

[0956] Human umbilical cord endothelial cells which are being maintainedin culture are transfected with the described plasmid using the methodknown to the skilled person (Lucibello et al., EMBO J. 14, 132 (1995)),and the quantity of β-glucuroridase which is produced by the endothelialcells, with and without the addition of cyclosporin A (0.01 to 1.0 μg/mlof culture medium), is measured using 4-methylumbelliferyl-β-glucuronideas a substrate.

[0957] For the purpose of checking the cell-cycle specificity,endothelial cells are synchronized in GO/GI by removing methionine for48 hours. The DNA content of the cells is determined in afluorescence-activated cell sorter following staining with Hoechst 33258(Hoechst AG, Frankfurt) (Lucibello et al., EMBO J. 14, 132 (1995)).

[0958] The following results are obtained:

[0959] In the absence of added cyclosporin A, it is not possible todetect any increase in β-glucuronidase in transfected endothelial cellsas compared with untransfected endothelial cells.

[0960] Following the addition of cyclosporin A, transfected endothelialcells express markedly more β-glucuronidase than do untransfectedendothelial cells.

[0961] Proliferating endothelial cells (DNA >2S) secrete markedly moreβ-glucuronidase than do endothelial cells which are synchronized inGO/GI (DNA=2S).

[0962] The self-enhancing expression system which has been describedresults in an expression of the structural gene β-glucuronidase which iscell cycle-dependent and which can be controlled by adding cyclosporinA.

[0963] The strength of the expression achieved by the novel,self-enhancing, pharmacologically controllable expression system isgreater than that achieved by the non-self-enhancing expression systemwhich was prepared in Example 1) in accordance with the diagram depictedin FIG. 14.

[0964] Priority application, 19651443.6 (Federal Republic of Germany),filed Dec. 12, 1996, including the specification, drawing, claims andabstract, is hereby incorporated by reference.

1 9 17 base pairs nucleic acid single linear other nucleic acid /desc =“binding sequence” 1 CGGACAACTG TTGACCG 17 7 amino acids amino acid<Unknown> linear peptide 2 Pro Lys Lys Lys Arg Lys Val 1 5 20 base pairsnucleic acid single linear other nucleic acid /desc = “binding sequence”3 TACTGTATGT ACATACAGTA 20 22 base pairs nucleic acid single linearother nucleic acid /desc = “binding sequence” 4 GAATTGTGAG CGCTCACAAT TC22 42 base pairs nucleic acid single linear other nucleic acid /desc =“binding sequence” 5 TCGAGTTTAC CACTCCCTAT CAGTGATAGA GAAAAGTGAA AG 4212 base pairs nucleic acid single linear other nucleic acid /desc =“binding sequence” 6 TAATGATGGG CG 12 26 base pairs nucleic acid singlelinear other nucleic acid /desc = “binding sequence” 7 GGAAGCAGACCACGTGGTCT GCTTCC 26 68 base pairs nucleic acid single linear DNA(genomic) 8 AGCAGGTGTT GGGAGGCAGC AGGTGTTGGG AGGCAGCAGG TGTTGGGAGGCAGCAGGTGT 60 TGGGAGGC 68 41 base pairs nucleic acid single linear DNA(genomic) 9 GGCCGATGGG CAGATAGAGG GGGCCGATGG GCAGATAGAG G 41

1. A nucleic acid construct that comprises: at least one firststructural gene that encodes an active compound; at least one secondstructural gene that encodes a transcription factor protein; and atleast one activation sequence comprised of at least one sequence thatbinds the transcription factor protein and at least one promotersequence; wherein each activation sequence activates the expression of astructural gene and the expression of the transcription factor protein.2. A nucleic acid construct according to claim 1, wherein an activationsequence is attached to the 5′ end of the first structural gene and anactivation sequence is attached to the 5′ end of the transcriptionfactor protein gene.
 3. A nucleic acid construct according to claim 1,that comprises two identical transcription factor protein bindingsequences and two non-identical promoters.
 4. A nucleic acid constructaccording to claim 1, further comprising an internal ribosomal entrysite wherein said internal ribosomal entry site participates inactivating expression of the transcription factor protein by theactivation sequence.
 5. A nucleic acid construct according to claim 1,further comprising: a nuclear export signal sequence appended to thefirst structural gene; a third promoter; and a nuclear export factorgene sequence.
 6. A nucleic acid construct according to claim 1, whereinat least two of the structural genes are mutually linked by an IRESsequence or by an activation sequence.
 7. A nucleic acid constructaccording to claim 1, wherein at least two transcription factor proteingenes are mutually linked by an IRES sequence or by an activationsequence.
 8. A nucleic acid construct according to claim 7, wherein saidtranscription factor protein genes are non-identical and transcriptionfactor proteins produced from said genes binds said transcription factorprotein binding sequences in the nucleic acid construct.
 9. A nucleicacid construct according to claim 1, further comprising apharmacological control module that comprises, in serial order: at leastone promoter; at least one fusion protein gene that comprises anactivation domain of a transcription factor protein, and a sequence thatbinds a coupling protein; at least one promoter; at least one fusionprotein gene wherein the fusion protein comprises a DNA-binding proteinand a protein that binds a coupling substance; and at least oneactivation sequence that comprises a site for the DNA-binding protein.10. A nucleic acid construct that comprises: at least one firststructural gene that encodes an active compound; at least one secondstructural gene that encodes a transcription factor protein; and atleast one activation sequence comprised of at least one sequence thatbinds said transcription factor protein and at least one pharmacologicalcontrol module that comprises, in serial order, at least one promoter,at least one fusion protein gene coding for an activation domain of atranscription factor protein and coding for a coupling substanceprotein, at least one promoter, at least one fusion protein gene codingfor a DNA-binding protein and coding for a second coupling substanceprotein, and at least one activation sequence that comprises a site forthe DNA-binding protein wherein each activation sequence activates theexpression of a structural gene and the expression of the transcriptionfactor protein.
 11. A nucleic acid construct that comprises: at leastone first structural gene that encodes an active compound; at least onesecond structural gene that encodes at least one first fusion proteinthat comprises an activation domain of a transcription factor protein,and a sequence that binds a coupling substance; at least one thirdstructural gene that encodes at least one second fusion protein thatcomprises a protein that binds a coupling substance and a DNA-bindingprotein; at least one activation sequence comprised of at least onesequence that binds said second fusion protein coupled to said firstfusion protein by a coupling substance and at least one promotersequence; wherein each activation sequence activates the expression ofat least one of said structural genes.
 12. A nucleic acid constructaccording to claim 1, wherein said activation sequence comprises asequence for binding a transcription factor protein, the sequenceselected from the group consisting of the Gal4 protein gene, the LexAprotein gene, the Lac I repressor protein gene, the tetracyclinrepressor protein gene, and the ZFHD-1 protein gene; a promoter sequenceselected from the group consisting of the basal c-fos promoter incombination with the HSV-1 VPI6 transactivation domain, the U2 sn RNApromoter in combination with a sequence of the Oct-2 activation domain,and the HSV TK promoter; and a transcription factor protein geneselected from the group consisting of the DNA-binding domain of the Gal4protein, the DNA-binding domain of the LexA protein, the Lac I repressorprotein gene, the tetracycline repressor protein gene and the ZFHD1protein gene.
 13. A nucleic acid construct according to claim 12,wherein said transcription factor protein comprises the SV40 nuclearlocalization signal and the HSV-1 VP16 acid transactivation domain. 14.A nucleic acid construct according to claim 9, wherein saidpharmacological control module comprises: a protein coding sequenceselected from the group consisting of the herpes virus VP16transactivation domain, the p65 subunit of the NF-KB transcriptionfactor, and the Oct-2 N-terminal glutamine-rich domain which is directlyor indirectly linked to the Oct-2 C-terminal proline-rich Oct-2 domain;a protein coding sequence selected from the group consisting of theDNA-binding domain of the Gal4 protein, the DNA-binding domain of theLexA protein, the Lac I repressor protein, the tetracycline repressorprotein, and the ZFHD1 protein; and at least one nucleic acid sequencethat binds a protein selected from the group consisting of the Gal4protein, the LexA protein, the Lac I repressor protein, the tetracyclinerepressor protein, and the ZFHD1 protein; wherein the nucleic acidsequence that binds a protein is linked to a promoter selected from thegroup consisting of the basal SV40 promoter, the c-fos promoter incombination with the HSV-1 VP16 transactivation domain, the U2 sn RNApromoter in combination with the HSV-1 VP16 transactivation domain orwith at least a sequence of the Oct-2 activation domain, and the HSV TKpromoter.
 15. A nucleic acid construct according to claim 14, furthercomprising an SV40 nuclear localization signal and an HSV-1 VP16 acidtransactivation domain wherein the SV40 nuclear localization signal andHSV-1 VP16 acid transactivation domain are present in a gene thatencodes a fusion protein.
 16. A nucleic acid construct according toclaim 9 wherein at least one fusion protein comprises an antibody orantibody fragment.
 17. A nucleic acid construct according to claim 16,wherein said antibody fragment comprises a single-chain Fv fragmenthaving a variable chain and a light chain where the variable and lightchains are linked covalently by a short peptide sequence.
 18. A nucleicacid construct according to claim 9, wherein at least one fusion proteincomprises a binding domain of a naturally occurring protein.
 19. Anucleic acid construct according to claim 1, wherein at least onepromoter is selected from the group consisting of RNA polymerase III,RNA polymerase II, CMV promoter and enhancer, SV40 promoter, an HBVpromoter, an HCV promoter, an HSV promoter, an HPV promoter, an EBVpromoter, an HTLV promoter, an HIV promoter, an cdc25C promoter, acyclin A promoter, a cdc2 promoter, a bmyb promoter, a DHFR promoter andan E2F-1 promoter.
 20. A nucleic acid construct according to claim 5,wherein the nuclear export signal and the corresponding nuclear exportfactor are selected from a rev-responsive element/rev protein of aretrovirus selected from the group consisting of HIV-1, HIV-2, HTLV-1and HBV.
 21. A nucleic acid construct according to claim 1, wherein thestructural gene encodes a compound selected from the group consisting ofinhibitors of cell proliferation, cytostatic or cytotoxic proteins,enzymes for cleaving prodrugs, antibodies, fusion proteins betweenantibody fragments and other proteins, cytokines, growth factors,hormones, receptors for cytokines and growth factors, cytokineantagonists, inflammation inducers, coagulation-inducing factors,coagulation inhibitors, fibrinolysis-inducing proteins, angiogenesisinhibitors, angiogenesis factors, hypotensive peptides, blood plasmaproteins, insulin receptor, LDL receptor, enzymes whose absence leads tometabolic diseases or immunosuppression, viral antigens, bacterialantigens, parasitic antigens or tumour antigens, antiidiotype antibodiesfor these antigens, and a fusion protein derived from any combination ofthese.
 22. A nucleic acid construct according to claim 9, wherein atleast two—structural genes are mutually linked by an IRES sequence or byan activation sequence.
 23. A vector comprising the nucleic acidconstruct of claim
 1. 24. A pharmaceutical composition comprising anucleic acid construct of claim 1 and a pharmaceutically acceptablecarrier.
 25. A pharmaceutical composition according to claim 24, furthercomprising a coupling substance that binds to a fusion protein.
 26. Apharmaceutical composition according to claim 25, wherein said couplingsubstance penetrates cell membranes and enters cells.
 27. Apharmaceutical composition according to claim 25, wherein said couplingsubstance is selected from the group consisting of rapamycin, FK506,cyclosporin A, methotrexate, folic acid, retinoic acid, penicillin,4-hydroxy tamoxifen, tamoxifen, tetracycline, and atetracycline/isopropyl-β-D-thiogalactoside conjugate.
 28. A cell whichcomprises a nucleic acid construct as described in claim
 1. 29. Aprocess for preparing a nucleic acid construct as described in claim 1,comprising: linking a sequence that binds a transcription factor proteinto a promoter sequence to form an activation sequence; and linking theactivation sequence to at least one structural gene and to at least onegene that encodes a transcription factor protein.
 30. A method oftreating a disease in a patient comprising administering the nucleicacid construct of claim 1 to a patient in need thereof, wherein thenucleic acid construct is administered in an amount sufficient toameliorate the disease.
 31. A method for vaccination of a patientcomprising administering the nucleic acid construct of claim 1 to apatient in need thereof, wherein the nucleic acid construct isadministered in an amount sufficient to prevent the disease.
 32. Amethod for preparing a pharmaceutical composition comprising:transforming a cell with a DNA construct as described in claim 1;culturing the transformed cell to obtain multiple copies of the DNAconstruct; and purifying DNA from the cultured cells.